The use of isoenzymes in characterization of grapevines (Vitis vinifera, L.). Influence of the environment and time of sampling

SCIENTIA HORTICULTUP,~ ELSEVIER

Scientia Horticulturae 69 (1997) 145-155

The use of isoenzymes in characterization of grapevines( Vitis vinifera, L.). Influence of the environment and time of sampling J.B. Royo a, F. Cabello b, S. Miranda a, y. Gogorcena b, J. Gonzalez a, S. Moreno b, R. Itoiz a, J.M. Ortiz b a Departamento de Producci6n Agraria, Universidad P{Iblica de Navarra. Naz'arra, Spain b Departamento de Biologia Vegetal, Universidad Polit~cnica de Madrid, Madrid, Spain

Accepted 13 January 1997

Abstract Isoenzymes from grapevine woody stems and shoots were evaluated for their use in identification of varieties and clones. Plant extracts were separated by polyacrylamide gel electrophoresis. Isoenzyme analysis was carried out for esterases, peroxidases, catechol oxidase, glutamate oxalacetate transaminase and acid phosphatase. The plant material was grown and sampled at two localities in Spain, with different climatic conditions. Sampling was carried out bimonthly for two consecutive years in order to find out the influence of the environment and time of the year. Each isozyme system had a pattern defined by "fixed' bands, that were always present at both localities and during the resting period of the plant (fall-winter). Esterases had a pattern with very few 'fixed' bands. Catechol oxidase was the most polymorphic system, thus being highly discriminating. The studied isoenzyme systems gave differences among varieties, although not among clones of the same variety. The use of this methodology for identification of grapevine plant material is discussed. © 1997 Elsevier Science B.V. Keywords: Acid phosphatase; Catechol oxidase; Esterases; Glutamate oxalacetate transaminase; Identification;

Peroxidases

Abbreviations: AAT (aminotransferase); 6GDH (glutamate dehydrogenase); GOT isomerase); LAP (leucine aminopeptidase); (peroxidases); PGM (phosphoglucomutase);

ACP (acid phosphatase); CO (catechol oxidase); EST (esterases); (glutamate oxalacetate transaminase); GPI (glucose phosphate MDH (malate dehydrogenase); PEG (polyethylene glycol); PER PVPP (polyvinyl polypyrrolidone)

0304-4238/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. Pll S0304-4238(97)00007-1

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1. Introduction Identification of grapevine (Vitis vinifera L.) varieties and cultivars has usually been carried out by morphological and agronomical characteristics following descriptor lists prepared by international organizations (O.I.V., I.P.G.R.I., etc.). These characters are often influenced by the environment (Dettweiler-Miinch, 1993). This makes advisable the use of alternative methods less affected by the environment (Subden et al., 1987; Cal6 et al., 1989). In the last decade, isoenzymes have been used with this objective (Weeden et al., 1988; Parfitt, 1989; Eiras-Dias et al., 1989; Parfitt and Arulsekar, 1989; Waiters et al., 1989). More recently, DNA molecular markers have been studied for grapevine identification purposes (Bowers et al., 1993; Thomas and Scott, 1993; Ortiz et al., 1994; Moreno et al., 1995). One of the supposed advantages of the isozymes versus the morphological characters is their higher independence from the environment. However, the technique followed for the preparation and analysis of isozymes, and the plant organ used, may have some influence on the final results. This variability is highest when using leaf extracts, since the leaves support major physiological changes. It has been observed (Almgard and Chapham, 1975) that fungal toxins can alter the leaf peroxidases patterns of grasses. Zymograms may differ when obtained at different growing stages of the plant. Also different results can be obtained from greenhouse plants compared to those grown in the open. Zymograms can also vary depending on the location of the plants or the year of the study. Subden et al. (1987), using different tissues from the same plant or the same tissue at different stages or ages, observed changes on the banding patterns for AAT, MDH, PGM and EST. Cal6 et al. (1989) on their side, found that PGM and GPI in leaves were stable, while AAT and LAP were unstable. Seasonal variation resulting in a higher or lower number of bands in the isozyme patterns has been observed, depending on the plant organ and the isozyme system (Gogorcena et al., 1993; Royo et al., 1993), although a thorough study has not been carried out for the woody stem extracts. In order to adequately use the isozyme systems for grapevine identification, the incidence of the mentioned factors has to be studied. The objectives of the present work were: i) to compare the studied isozyme patterns at two different locations in order to detect the influence of the environment; ii) to analyze the extracts at different times of the year in order to decide the most adequate time of sampling; iii) to define the discriminant value of the zymograms for identification of varieties. The selection of the isozyme systems was based on the results obtained with different systems in respect to the number of polymorphic bands (Altube et al., 1991; Cabello et al., 1994; Royo et al., 1994; Cabello and Ortiz, 1995).

2. Material and methods

2.1. Plant mawrial Eight varieties of Vitis vinifera L., traditionally grown in Spain were used in this study (Table 1). In four of the varieties, we included two clones each for comparison

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Table 1 List of varieties and clones of Vitis cinifera L. used in the study Common name

Clones Code Origin

Garnacha

CZ 5.68 G68 'Universidad Pfiblicade Navarra' CG 5.28 G28

Tempranillo

ILl 51 RJ 43

Cabernet sauvignon ID 15 Viura 1D Chardonnay 1E

T51 'Centro de Investigaci6n y Desarrollo Agrario de la Rioja" T43 CID 'Estacidn de Viticultura y Enologla de Navarra' C15 VIU CHA

Malvar Airen Tinto fino

22-1-34 MAL 'Banco de Germoplasma de Vid de "El Encin" (Comunidad de Madrid)' 22-I-3 AIR 22-J-2 TFN

Bobal

55 22

B55 'Universidad Polit~cnica de Valencia' B22

purposes. Also, two cultivars ( ' T e m p r a n i l l o ' and 'Tinto F i n o ' ) which are believed to be synonymies of the same variety were included. The plants were propagated in the greenhouse from dormant hardwood cuttings taken from the same vinestock. Afterwards they were transferred to the open, both in Madrid and in Pamplona. The Thornthwaite classification of Madrid climate places it as semiarid (DB'2d), average temperature 14.1°C, rainfall 453 mm. Pamplona climate is classed as humid (B1B'Is), average temperature 12.4°C, rainfall 863 mm. Plants were grown by following the usual agricultural practices. Samples were taken at both localities from the beginning of the first rest period following transplant. 2.2. Plant organs and time o f sampling

Based on Subden et al. (1987) and in previous studies carried out at our laboratory, 10 cm long stems from the subapical region were sampled during the f a l l / w i n t e r 1992-1993 and 1993-1994, at 80% leaf drop, at the middle of the rest and at the beginning of sap activity. Further samplings were carried-out during the growing season 1993 at the ' G ' stage of Baggiolini, two weeks after bloom and 40 days after coiour break of the berries. A total of nine samples per variety and location were collected. The dates for sampling varied owing to the phenology. 2.3. Analytical methods

Isozyme analysis was carried out by electrophoresis in polyacrylamide gels. Extracts were prepared by homogenization in an electric grinder of 2 g of previously minced plant material in 10 ml of 0.5 M tris-citrate buffer at pH 8.0 with 10 m M cysteine and 5

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i

CO

ACP i

GOT i

PER

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149

mM ascorbic acid as antioxidants. For precipitation of the polyphenols 1% PEG 4000 and 15% PVPP were included. The protein fraction was separated by centrifugation at 10000 r.p,m, for 20 min. Gels were 1 mm thick with three layers: upper stacking gel (7.6 cm), upper resolving gel (1.3 cm) and lower resolving gel (7.6 cm), containing 4, 6.3 and 10% acrylamide, respectively. Gel buffers were 0.5 M tris-HCl at pH 8.0 for the first layer and 1.5 M tris-HCl at pH 8.8 for the two other layers. Electrode buffer was 0.025 M tris-glycine at pH 8.5. Electrophoretic separation was carried out at 2 mA per well for 3 h. An identical methodology was used at both laboratories in order to obtain comparable results. Plant extracts were kept at - 2 0 ° C until analysis, which was carried out within 10-15 days from sampling. The isozyme systems studied were: peroxidases (EC.I.10.3.1.); esterases (EC.3.1.1.1.); catechol oxidase (EC. 1.10.3.1.); glutamate oxalacetate transaminase (EC.2.6.1. l.); and acid phosphatase (EC.3.1.3.2.). Staining techniques were carried out according to Arulsekar and Parfitt (1986) for PER, Schwennesen et al. (1982) for CO and ACP, Sfi.nchez-Yrlamo (1992) for GOT, and Kuhns and Fretz (1978) for EST.

3. Results In both localities, woody stem extracts sampled during winter yielded sharper and more repeatable band patterns than extracts from stems sampled during the growing season (Fig. 1). Accordingly, only extracts from stems sampled during the resting period (fall/winter) were analyzed. In every case, two types of bands were detected: i) 'fixed' bands which were always present during the three analyses carried out in the fall-winter period, regardless of the locality or year; ii) 'variable' bands, that appeared erratically, only in some of the samplings, or in one of the years or localities. These variable bands were often very weak in intensity. Consequently, the comparison of the results was based on the 'fixed' bands. 3.1. Esterases

This system yielded a very high number of 'variable' bands. Out of a total of 18 bands, only 4 of them were of the 'fixed' type. The detailed study of the results from this system (data not presented) indicated that from the characterization point of view, is not advisable. 3.2. Catechol oxidase

In the CO system (Table 2) there were eight ~fixed' bands, (band numbers 1-6, 8 and 10). A total of four 'variable' bands were occasionally present (Table 2). In most cases,

Fig. 1. Electrophoretic separation of CO (catechol oxidase), ACP (acid phosphatase), GOT (glutamate oxalacetate transaminase) and PER (peroxidases), from woody stem extracts of 'T51' from Pamplona, "VIU' from Madrid, "CHA" from Pamplona and "C15' from Madrid, respectively. 1 and 7 are leaf drop; 2 and 8 are middle rest: 3 and 9 are sap activity: 4 is Baggiolini 'G" stage; 5 is after bloom: 6 is colour break. 1-3 are from the first year of study: 7-9 are from the second year of study. See text for details.

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Table 2 Fixed (F) bands and frequencies of the variable bands (xM, yP) " obtained from the 12 analyses of cathecol oxidase (CO) from extracts of the fall-winter woody stems Band no.

AIR b

B22 B55

CHA

F

(0M, 1P)

CID C15

G28 G68

1

2 3 4 5 6 (7) 8 (9) 10 (12) (14)

F

F F

F F (1M,2P) (IM,OP)

(2M, 1P) ( 1M,OP) F (IM,1P) (IM.0P)

F F (0M,1P)

F F (0M, 1P) (OM, 1 P) F (0M,1P) F (IM,1P) (0M,2P)

MAL

TFN T43,T51

VIU

F

F

F

F

F

F

F

F

(0M,3P)

F

F

F F F F (OM, 1P) F (0M, IP) (2M,1P)

F

F

(0M,3P)

xM. vP is number of times that this band was present in Madrid (M) and in Pamplona (P). b See Table 1 for cultivar codes. a

t h e s e v a r i a b l e b a n d s w e r e faint, in c o n t r a s t to the ' f i x e d ' b a n d s that w e r e strong. O n l y o n e o f the ' f i x e d ' b a n d s ( b a n d 8) w a s p r e s e n t in all s a m p l e s . T h e z y m o g r a m s o f the s t u d i e d varieties s h o w e d a total o f s e v e n d i f f e r e n t p a t t e r n s b a s e d o n the ' f i x e d ' b a n d s (Fig. 2). E a c h o n e o f the varieties h a d a d i f f e r e n t pattern, w i t h the e x c e p t i o n o f ' B o b a l ' a n d ' V i u r a ' , that s h o w e d the s a m e one. ' T i n t o F i n o ' a n d ' T e m p r a n i l l o ' , w h i c h are t w o s y n o n y m i e s o f the s a m e cultivar, h a d the s a m e z y m o g r a m .

CO

ACP A

B

A

B

C

D

E

F

G

1

m

2

- -

4 - s 6

- -

- -

- -

m

GOT

A

PER

B

A

B

C

2

5 6

Fig. 2. Zymogram types (see Table 6) obtained with the 'fixed' bands for each of the studied isozyme systems. Band numbers correspond to those in Table 2Table 3Table 4Table 5.

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151

Table 3 Fixed (F) bands and frequencies of the variable bands (xM, yP) a obtained from the 12 analyses of acid phosphatase (ACP) from extracts of the fall-winter woody stems Band no.

AIR b

B22 B55

CHA

C1D C15

G28 G68

MAL

TFN T43,T51

1

F

F

F

F

F

F

F

F

2 3 4 5 (6) 7 (8) (9) 10

F F F F (2M, IP) F (1M,0P) (0M,3P) F

F F

F F

F F

F F

F F

F (2M, IP) F (2M,0P) (0M,3P) F

F (2M,1P) F (2M,0P) (0M,3P) F

F (2M,0P) F (2M,OP) (OM,3P) F

F (2M.1P) F (2M,OP) (OM,3P) F

F F F F (2M, IP) F (IM,0P) (OM,3P) F

F F F F (2M, IP) F (2M,OP) (0M,3P) F

F (2M. IP) F (2M,0P) (0M,3P) F

VIU

xM, yP is the number of times that this band was present in Madrid (M) and in Pamplona (P). See Table 1 for cultivar codes.

3.3. Acid phosphatase Table 3 summarizes the results for this system. Bands 6, 8 and 9 were variable and only present in some cases. Out of the seven 'fixed' bands detected, only band 4 was polymorpbic for the studied samples. Consequently, this system provides only two zymogram patterns (Fig. 2), one for 'Malvar', 'Airrn' and 'Viura', and the other for the rest of varieties.

3.4. Glutamate oxalacetate transaminase In this system (Table 4), four 'fixed' bands were present in some of the samples. Bands 1 and 2 were always present in all cultivars. Bands 3 and 4 were 'fixed' in some cultivars and 'variable' in others. Bands 5 and 6, when present, were 'variable'. As in the other systems, the 'variable' bands were always faint in intensity. Three different patterns were obtained (Fig. 2), one for 'Air~n', another one for 'Cabernet Sauvignon' and a third one shared by the other cultivars.

Table 4 Fixed (F) bands and frequencies of the variable bands ( x M , y P ) a obtained from the 12 analyses of glutamate oxalacetate transaminase (GOT) from extracts of the fall-winter woody stems Band no.

AIRb

B22 B55

CHA

CIDCI5

G28 G68

MAL

TFN T43,T51

VIU

1

F

F

F

F

F

F

F

F

2 3 4 (5) (6)

F (0M. IP) F (4M,3P)

F F F (3M,3P) (0M, IP)

F F F (2M,3P) (0M,1P)

F (0M,2P) (0M,3P)

F F F (2M,3P) (0M, IP)

F F F (2M,3P) (0M, IP)

F F F (2M,3P) (0M,It i)

F F F (2M,3P) (0M, IP)

xM, yP is the number of times that this band was present in Madrid (M) and in Pamplona (P). h See Table 1 for cultivar codes.

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Table 5 Fixed (F) bands and frequencies of the variable bands (xM vP)a obtained from the 12 analyses of peroxidases (PER) from extracts of the fall-winter woody stems Band no.

AIR b

B22 1355 CHA

1

F

F

2 3 (4) 5 6~ 7c (8)

F F (3M,0P)

F F (3M,0P) F F F

F F

C1DC15

G28 G68

MAL

TFN T43,T51

VIU

F F F (3M,0P)

F F (3M,0P)

F F

F F

F F (3M,OP)

F F (3M,0P)

F F (3M,0P)

F F F (3M,0P)

F F (0M,3P)

F F

F F

F F

xM._vP is the number of times that this band was present in Madrid (M) and in Pamplona (P). h See Table 1 for cultivar codes. Bands that frequently appear as double. a

3.5. Peroxidases T a b l e 5 s h o w s the results for this system. Six ' f i x e d ' b a n d s were present, b a n d s 1 a n d 5 b e i n g p o l y m o r p h i c . B a n d 4 w a s ' v a r i a b l e ' in all s a m p l e s , w h i l e b a n d 8 only o c c u r r e d in s o m e cases in ' C h a r d o n n a y ' . T h r e e d i f f e r e n t z y m o g r a m p a t t e r n s w e r e o b s e r v e d (Fig. 2), o n e for ' B o b a l ' , a n o t h e r o n e for ' A i r ~ n ' , ' M a l v a r ' , ' T i n t o F i n o ' a n d ' T e m p r a n i l l o ' a n d a third o n e for the rest.

3.6. Identification o f grapeuine cultie, ars T a b l e 6 s h o w s the d i f f e r e n t z y m o g r a m p a t t e r n s o b t a i n e d w i t h the studied i s o z y m e s y s t e m s . E a c h c o l u m n in this T a b l e has a d i f f e r e n t c o m b i n a t i o n o f patterns, u n i q u e l y i d e n t i f y i n g the varieties u n d e r study. M o r e o v e r , b y u s i n g the C O s y s t e m t o g e t h e r w i t h e i t h e r A C P or P E R , e v e r y variety c a n b e identified. T h e t w o s y n o n y m i e s ( ' T i n t o F i n o ' a n d ' T e m p r a n i l l o ' ) a l w a y s y i e l d e d the s a m e pattern. N o n e o f the s t u d i e d i s o z y m e s y s t e m s w a s able to d e t e c t d i f f e r e n c e s a m o n g the c l o n e s o f the s a m e variety.

Table 6 Pattern types of the studied varieties Isozyme system ~

AIR b

B22 B55

CHA

C 1D C 15

G28 G68

MAL

TFN T43,T51

VIU

CO ACP GOT PER

Ac B B

B A A

C A A

D A C

E A A

F B A

G A A

B B A

A

B

C

C

C

A

A

C

CO, catechol oxidase; ACP, acid phosphatases; GOT, glutamate oxalacetate transaminases; PER, peroxidases. h See Table 1 for cultivar codes. See schematic pattern types in Fig. 2.

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4. Discussion During fall/winter we obtained a good resolution of reproducible bands, while during the growing season, a variable number of erratic bands was often present. For this reason, the study of the zymogram profiles was carried out with the results of the fall-winter period. Woody stems were used based on previous results obtained in our laboratory (Altube et al., 1991; Gogorcena et al., 1993; Cabello and Ortiz, 1995). Polyacrylamide gels are more inert and have a more uniform molecular sieving effect than starch gels (Hames and Rickwood, 1981; Weeden, 1988), and the results obtained were quite reproducible. Furthermore, the use of discontinuous gels causes a concentration of the proteins before reaching the resolution gel. For these reasons, added to the higher transparency of the support, we could detect more bands than reported using the starch gels (Chrambach and Roadbard, 1971). The catechol oxidase, peroxidases, acid phosphatase and esterase systems, enzymes that are active on different substrates, are more polymorphic and hence of a higher value for identification purposes than enzymes that need specific substrates (PGM, GPI, GOT, 6GDH . . . . ). However, the first group of enzymes, seem to be more influenced by the environment (Gillespie and Kojima, 1968; Gillespie and Langley, 1974; Weeden, 1988). As previously stated by Eiras-Dias et al. (1989) and Subden et al. (1987), the esterase zymograms were highly variable, even during the rest period. Consequently, this system should not be used for identification purposes. Catechol oxidases have been so far not much used for identification purposes. Schwennesen et al. (1982) and Cabello and Ortiz (1995) studied this system doing the sampling at the beginning of winter. Walters et al. (1989), using starch gels, obtained reproducible results from samplings in April and July, although the faint bands were not clearly visible on this support. Altube et al. (1991) obtained reproducible results in acrylamide gels, from woody stem extracts sampled during the resting period. Peroxidases are one of the most used systems for identification, although few studies have been carried out to evaluate their stability. Reproducible patterns were obtained by Altube et al. (1991) using woody stem extracts sampled in winter, even using plants from different environments (Bachmann and Blaich, 1988). The results are not reproducible during the growing season (Subden et al., 1987). The glutamate oxalacetate transaminase was also unstable during the growing season. Although Benin et al. (1988) obtained reproducible results during the growing season by using extracts from plants grown in controlled conditions, other authors stated the variability of the results during the same period (Eiras-Dias et al., 1989; Parfitt and Arulsekar, 1989; Cal~) et al., 1989). The constancy of the results during the resting period for woody stem extracts has been obtained by other authors (Altube et al., 1991 ; Walker and Boursiquot, 1992; Subden et al., 1987). With respect to the acid phosphatases, Walters et al. (1989), and Subden et al. (1987), using starch gels, and Benin et al. (1988) with polyacrilamide, obtained reproducible results during the whole cycle. Altube et al. (1991) and Walker and Boursiquot (1992), also reported the reproducibility of the results during the rest period. The discriminant capacity of each of the studied systems was different. Catechol oxidase was the most polymorphic system, being able to distinguish most of the studied

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varieties. Consequently, although it has not been broadly used up to the present, this system seems highly promising for identification purposes. None of the studied systems were able to distinguish among clones of the same variety. Similar results have been obtained in other studies with isozymes (Calb et al., 1989; Gogorcena et al., 1993). Recent studies carried out with molecular markers (Moreno et al., 1995) indicate the possibility of clonal distinction in some cases. As a conclusion, for the identification of grapevine cultivars we recommend the use of woody stem extracts, sampled during the winter period (between leaf fall and start of sap activity). For comparison purposes, a standardized methodology should be followed and the definition of the profiles based on the 'fixed' bands. Two or three repetitions of samplings and analysis are needed for the accurate obtention of the zymogram patterns.

Acknowledgements This study has been carried out with financial support provided by 'Comisi6n Interministerial de Ciencia y Tecnologia' (C.I.C.Y.T.), Project AGR 0070-91-C01-C02.

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