Differential gene expression for isozymes in somatic mutants of Vitis vinifera L. (Vitaceae)

Biochemical Systematics and Ecology 33 (2005) 691e703 www.elsevier.com/locate/biochemsyseco

Differential gene expression for isozymes in somatic mutants of Vitis vinifera L. (Vitaceae) Sandra Aparecida de Oliveira Collet, Marcos Andre´ Collet, Maria de Fa´tima P.S. Machado * Department of Cell Biology and Genetics, State University of Maringa´, Avenida Colombo, 5790, 87020-900 Maringa´, PR., Brazil Received 23 April 2004; accepted 24 December 2004

Abstract Isozyme electrophoresis was used as a method to provide a measure of relationship among Italia, Rubi, Benitaka, and Brasil cv of Vitis vinifera traditionally grown in Marialva, a town in the northwestern region of the state of Parana´, southern Brazil. No allelic variation was observed for esterase (EST), malate dehydrogenase (MDH), peroxidase (POD), glutamate dehydrogenase (GTDH), alkaline phosphatase (AKP), acid phosphatase (ACP), and aspartate amino transferase (AAT). Tissue specific and variation in staining intensity of EST, MDH, POD, and GTDH isozymes indicate differential gene expression in colour grape varieties. Regulatory genes may be operative in determining the number of molecules of enzymes in a cell and determining the berry skin polymorphism in four cultivars. Change frequency for berry skin colour suggest the occurrence of somatic crossing-over in naturally cultivated plants and a periclinal chimerism in Brasil cv. The four grape colour cultivars seem to be clones of the same cultivar. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Grapes; Isozymes; Somatic mutations; Somatic crossing-over; Polymorphism

* Corresponding author. E-mail address: [email protected] (M.F.P.S. Machado). 0305-1978/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2004.12.016

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1. Introduction Somatic mutations are particularly important for vegetative-propagated plant species since a mutant cell may be the progenitor of a population of identical mutant cells. A mutant sector may consist of a stem or leaf cutting that includes a mutant somatic sector and may be vegetatively propagated (Griffiths et al., 1996). Somatic mutations in grapes are relatively frequent events, associated with economically important morphological characteristics and have been accountable for the start of new varieties (Camargo, 1994; Franks et al., 2002; Vouillamoz et al., 2003). Somatic mutations in Italia cultivars seem to result in two kinds of Vitis vinifera, one with a light rosy red colour of the berry skin and another with a more intense and uniform rosy red colour. Mutation occurred on a side branch of vegetativepropagated Italia cultivars and resulted in Rubi cultivars (Kishino and Mashima, 1980). Another mutation also arose on one side branch of Italia cultivars, starting Benitaka cultivars (Sousa, 1996). Somatic mutation has also been reported as responsible for the origin of Brasil cultivars from one Benitaka cultivar side branch (Gonc¸alves, 1995). Black and red colours have been detected in the berry skin and pulp flesh, respectively, of Brasil cultivars. The colour of red and black grapes results from the accumulation of anthocyanins usually located in berry skin only. Control of anthocyanin pathway genes in grape berries has been studied and the anthocyanin biosynthetic pathway expression of seven genes determined (Boss et al., 1996). The appearance of anthocyanins in grape berry skin at the onset of ripening coincides with increased expression of each of the genes encoding biosynthetic enzymes in the pathway. The involvement of regulatory genes has been suggested. Regulatory genes involved in anthocyanins biosynthesis have been studied in maize (Martin and Gerats, 1993; Ludwing and Wessler, 1990), snapdragon flowers (Almeida et al., 1989; Martin et al., 1991; Martin and Gerats, 1993), and petunia (Beld et al., 1989; Quattrocchio et al., 1993). Regulatory gene mutants have shown a differential effect in anthocyanin gene expression in the most studied species (Boss et al., 1996). In current study isozyme electrophoresis was used as a method to determine the genetic variability in constitutive genes of the Italia, Rubi, Benitaka, and Brasil cultivars of V. vinifera. Isozyme analysis is of significant value to provide a measure of relationship among the four grape cultivars.

2. Material and methods The V. vinifera L. Italia cv (Piro´vano 65) was introduced in the state of Sa˜o Paulo, Brazil, in 1920, although its culture started only in the 1962s in the north of the southern state of Parana´ (Camargo, 1994). Somatic mutation occurring in side branches of the Italia and Benitaka cultivars (Fig. 1) was responsible for the start of four actually known cultivars (‘Italia’, ‘Rubi’, ‘Benitaka’, and ‘Brasil’) which are traditionally grown in the rural region of Marialva, a town in the northwestern region of the state of Parana´, southern Brazil (Fig. 2).

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ITÁLIA 1972 Santa Mariana region (Northeast of Paraná)

RUBI

1988 Floraí region (Northwest of Paraná)

BENITAKA

1991 Floraí region (Northwest of Paraná)

BRASIL

Fig. 1. Origin of the Rubi and Benitaka cultivars from Italia (1972 in Santa Marina region and 1988 in Florai region, respectively), and Brasil cv from Benitaka cv (1991 in Florai region).

Five thousand samples of randomly selected vines from 14 commercial Italia cv vineyards cultivated in Marialva were scored for the occurrence of mutant sectors in berry skin colour, at 2-week intervals for each vineyard, from 1991 to 2002. Other randomly selected samples of 11,000, 4500, and 700 vines of Rubi, Benitaka, and Brasil cultivars, respectively, were also scored for changes in berry skin colour. Electrophoresis evaluations were carried out in NovembereDecember 2002 for analysis of the malate dehydrogenase (MDH; EC 1.1.1.37), esterase (EST; EC 3.1.1.1), acid phosphatase (ACP; 3.1.3.2), alkaline phosphatase (ALP; EC 3.1.3.1), aspartate amino transferase (AAT; EC 2.6.1.1), glutamate dehydrogenase (GTDH; EC 1.4.1.2), and peroxidase (POD; 1.11.7.1). Samples of seeds, young leaves, tendrils, floral buds, and skin of each grape cultivar (Table 1) were individually homogenized using 0.1 M TriseHCl buffer, pH 8.5, 4% PVP-40 (polyvinylpyrrolydone), 1% bmercaptoethanol solution, 0.2% EDTA, 0.2% ascorbic acid, 0.38% sodium borate, 0.38% sodium chloride, 4% PEG (polyethyleneglycol), 2.5% DIECA (diethylethanocarbazole), and 1% Triton X-100 (Schaefer, 1971 modified). The samples were prepared by homogenizing each 100 mg tissue separately with a glass rod in a microcentrifuge tube containing 60 mL of cold extraction solution in an ice bath. The homogenates were centrifuged at 25,000 rpm for 30 min at 4  C in a Sorval 3K-30 centrifuge (Sigma). Supernatants were absorbed with Whatman no. 3 paper strips (5 ! 6 mm), and these were vertically inserted into a 16% starch gel (Penetrose-50Ò), prepared in 0.01 M Tris and 0.0028 M citric acid buffer, pH 7.5 for analysis of the EST, ACP, and PER isozymes. Tris (0.1 M) and citric acid (0.028 M), pH 7.5, were used in the electrode chambers. Electrophoresis was carried out at low temperature (10  C), for 4e5 h, at 38 mA (8.5 V/cm of gel). MDH, GTDH, ALP, and AAT isozymes were analyzed using starch gels prepared in 0.05 M histidine, 0.0014 M EDTA, pH 7.0, adjusted with 1.0 M Tris, and the electrode buffer consisted of 0.125 M Tris, pH 7.0, adjusted with 1.0 M citric acid. Electrophoresis was carried out at 10  C, for 15e16 h, at 2.5 V/cm of gel. The esterase isozymes were visualized by procedures originally described by Resende et al. (2000) and MDH isozymes were stained with the reaction mixture reported by Machado et al. (1993). A staining solution containing 50 mL 0.1 M TriseHCl buffer, pH 8.0, 300 mg glutamic acid, 6 mg NAD (b-nicotinamide adenine dinucleotide), 0.5 mL MTT (thiazolyl blue; 5 mg/mL), and 0.5 mL PMS (phenazine methosulfate; 5 mg/mL) was used to visualize GTDH isozymes. ACP isozymes were

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Fig. 2. Localities in the Marialva region, state of Parana´, southern region of Brazil, where samples of Vitis vinifera cultivars (Italia, Rubi, Benitaka, and Brasil) have been maintained by vegetative propagation. Producers: (a) Jaime Moreno; (b) Cezar Martini; (c) Jose´ Zanutto; (d) Ame´lia Yamanaka; (e) Benı´ cio Bonifa´cio; (f) Antonio Peres; (g) Se´rgio Ferracin; (h) Aure´lio Boro; (i) Lae´rcio de Pintor; (j) Idewalter Hungare; (k) Emı´ lio Na´poli Sobrinho; (l) Aparecido Beloti; (m) Luiz Ricieri; (n) Elena Mestre; (o) Jose´ Faria.

visualized after 30 min on a gel incubated at 37  C with 50 mL 0.1 M sodium acetate buffer, pH 5.0, 1.5 mL 1% a-naphthyl phosphate, and 25 mg fast blue RR salt, and ALP isozymes were incubated with 50 mL 0.05 M TriseHCl buffer, pH 8.5, 1.5 mL 1% a-naphthyl phosphate, 0.5 mL 1.0 M MgCl2, 0.5 mL 1.0 M MnCl2, and 25 mg fast blue RR salt. For POD isozymes’ visualization the gel was incubated for 15 min with 25 mL 1.0 M sodium acetate buffer, pH 4.7, 25 mL methanol, and 50 mg benzidine. Then, 2 mL H2O2 was added to the staining mixture and the preparation was maintained at 37  C until the time of POD isozymes detection. AAT isozymes were visualized after 30 min on a gel incubated at 37  C with 100 mL distilled water pH 8.0, adjusted with NaOH, 250 mg aspartic acid and 150 mg a-cetoglutaric acid, and 150 mg fast blue BB salt.

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Table 1 Number of plants and seed, leaf, tendril, floral buds, and skin tissues of the Italia, Rubi, Benitaka, and Brasil cultiavars for each enzymatic system Enzyme

EST

Tissue

Italia

Rubi

Benitaka

Brasil

Seed Skin Leaf Tendril Floral buds

11 8 76 13 17 76

11 10 80 13 23 80

14 13 38 11 11 38

12 12 17 12 16 17

Seed Skin Leaf Tendril Floral buds

15 11 62 10 19 62

17 10 83 11 12 83

21 12 43 9 11 43

11 12 22 11 10 22

Seed Skin Leaf Tendril Floral buds

12 11 39 10 10 39

12 11 61 11 10 61

12 11 26 9 12 26

10 10 15 11 12 15

Seed Skin Leaf Tendril Floral buds

14 12 42 10 10 42

14 12 44 11 10 44

11 11 15 9 12 15

11 11 9 9 9 9

Leaf Leaf Leaf Tendril Floral buds

36 10 65 15 5 65

41 14 91 19 5 91

16 8 12 10 10 12

10 4 21 10 6 21

Total of plants MDH

Total of plants POD

Total of plants GTDH

Total of plants ALP AAT ACP

Cultivars

Total of plants

3. Results and discussion The esterase isozyme patterns in seed, skin, leaf, tendril, and floral bud tissues of the four V. vinifera cultivars recorded with 4-methylumbelliferyl acetate and a-naphthyl acetate showed 14 esterase isozymes. Substrate preference for 4-methylumbelliferyl acetate and a-naphthyl acetate and tissue specificity for esterase isozymes has been reported (Table 2), however, no allelic variation was detected in plants of Italia, Rubi, Benitaka, and Brasil cultivars. Fig. 3A shows the esterase pattern detected in leaves of four grape cultivars. Tissue specificity was also detected for MDH, POD, and GTDH isozymes (Table 3) but no variation was observed in the isozyme patterns in seed, skin, leaf, and floral

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buds of the Italia, Rubi, Benitaka, and Brasil cultivars. Monomorphic patterns for MDH, POD, GTDH, ALP, and ACP isozymes are shown in Fig. 3BeF, respectively. Malate dehydrogenase exhibited a high molecular heterogeneity in berries and other tissues (Taureilles et al., 1992). These authors have suggested that the multiplicity of MDH forms could be correlated with numerous functions of this ubiquitous enzyme. According to the pattern of changes in mRNA transcript level, mtMDH is involved in both synthesis and degradation of malate in the berry (Or et al., 2000). In Italia, Rubi, Benitaka, and Brasil cultivars, the faster anodal region (MDH-6/ MDH-7/MDH-8 isozymes) can be considered to be the cytoplasmatic MDH isozyme (sMDH), the region of intermediate activity (MDH-3/MDH-4/MDH-5 isozymes) can be considered to be of mitochondrial origin (mtMDH), and slower region (MDH-1/ MDH-2 isozymes) can be considered to be a microbody isozyme (mbMDH), in agreement with data reported for most plants (Newton, 1983; Machado et al., 1993; Jorge et al., 1997; Orasmo and Machado, 2003). In seeds, the sMDH isozymes (MDH-6/MDH-7/MDH-8) and the mtMDH isozymes (MDH-3/MDH-4/MDH-5) are formed by three regularly spaced bands. Unchanged mtMDH and sMDH isozyme phenotypes formed by three regularly spaced bands with the intermediate band frequently being more intensely stained than the other bands, reflect an enzymedimeric structure (Pasteur et al., 1988) produced by two mtMDH and sMDH genes. MDH isozymes are recognized as dimers in the majority of plants (Douce and Neuburger, 1990). In leaves and floral buds the faster anodal MDH isozymes also show the heterodimeric pattern. Weeden et al. (1988) suggested four loci code for MDH isozymes in seedlings and young leaves. However, the three-banded phenotype

Table 2 Substrate preference for 4-methylumbelliferyl acetate (4-MUA) and a-naphthyl acetate (a-NA) and tissue specificity for esterase isozymes in plants of Italia, Rubi, Benitaka, and Brasil cultivars Isozyme

EST-1 EST-2 EST-3 EST-4 EST-5 EST-6 EST-7 EST-8 EST-9 EST-10 EST-11 EST-12 EST-13 EST-14

Seed

Skin

Leaf

Tendril

Floral bud

4-MUA

a-NA

4-MUA

a-NA

4-MUA

a-NA

4-MUA

a-NA

4-MUA

a-NA

C C ÿ ÿ ÿ ÿ ÿ C C C ÿ ÿ C C

C C C C ÿ C C ÿ ÿ ÿ ÿ ÿ ÿ ÿ

C C ÿ ÿ ÿ ÿ ÿ C C C ÿ ÿ ÿ ÿ

C C ÿ ÿ C ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ

C C ÿ ÿ ÿ ÿ ÿ C C C C C ÿ ÿ

C C ÿ ÿ ÿ ÿ ÿ C C C C C ÿ ÿ

C C ÿ ÿ ÿ ÿ ÿ C C C C C ÿ ÿ

C C ÿ ÿ ÿ ÿ ÿ C C C C C ÿ ÿ

C C ÿ ÿ ÿ ÿ ÿ C C C C C ÿ ÿ

C C ÿ ÿ ÿ ÿ ÿ ÿ C C ÿ ÿ ÿ ÿ

(C) Present and (ÿ) absent isozyme.

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Fig. 3. Isozyme patterns for young leaves of Italia (samples 1, 2, 3), Rubi (samples 4, 5, 6), Benitaka (samples 7, 8, 9), and Brasil (samples 10, 11, 12) cultivars of Vitis vinifera. Isozymes: esterase (A), malate dehydrogenase (B), peroxidase (C), glutamate dehydrogenase (D), alkaline phosphatase (E), and acid phosphatase (F).

for the dimeric mtMDH and sMDH isozymes may be representing heterozygosity at both loci since V. vinifera is an interspecific hybrid origin (Muscat of Hamburg ! Bicane). Because grape cultivars have been maintained as clones, heterozygous phenotypes would be expected. Restriction to homodimers formation or preferential association of mtMDH and sMDH isozymes heterodimers seems to be occurring in skin tissues and in mtMDH isozymes of leaves and floral buds (Fig. 3B). The use of isozymes for identification of grape cultivars and clones have been recommended (Sanches-Escribano et al., 1998), however, the most of the studied enzyme systems have not been able to detect differences among the clones of the same variety (Royo et al., 1997; Eiras-Dias et al., 1989). POD isozymes have been reported as one of the most used system for grape cultivars identification (Altube et al., 1991; Royo et al., 1997) and ACP isozymes staining intensity have been taxonomically informative for comparison between of the hybrids (crossing of the species Vitis riparia, Vitis rupestris and Vitis berlandieri; Tedesco et al., 1997). In Italia, Rubi,

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Table 3 Tissue specificity for malate dehydrogenase (MDH), peroxidase (POD), acid phosphatase (ACP), alkaline phosphatase (ALP), aspartate amino transferase (AAT), and glutamate dehydrogenase (GTDH) isozymes in plants of Italia, Rubi, Benitaka, and Brasil cultivars

MDH-1 MDH-2 MDH-3 MDH-4 MDH-5 MDH-6 MDH-7 MDH-8 POD-1 POD-2 GTDH-1 GTDH-2 GTDH-3 GTDH-4 ALP-1 ALP-2 AAT-1 AAT-2 ACP-1 ACP-2 ACP-3

Seed

Skin

Leaf

Tendril

Floral bud

ÿ ÿ C C C C C C C C C C ÿ ÿ nd nd nd nd nd nd nd

ÿ ÿ ÿ C C C C C C ÿ ÿ C ÿ ÿ nd nd nd nd nd nd nd

C C ÿ C ÿ C C C C C C C C C C C C C C C C

ÿ ÿ ÿ C ÿ C C C C C C C C C nd nd nd nd C C C

ÿ ÿ ÿ C C C C C C ÿ ÿ C ÿ ÿ nd nd nd nd C C C

(C) Present and (ÿ) absent isozyme; nd: not determined.

Benitaka, and Brasil cultivars, ACP-4 and ACP-5 isozymes were weakly stained; it was not detected in all samples of leaves (Fig. 3F), therefore, were not computed for the comparative electrophoretic analysis of different tissues and cultivars. On the other hand, isozymes were used to measure the genetic stability of clones and of introduced long-established cultivars (V. vinifera cv Chardonnay; Subden et al., 1992). The lack of isozyme polymorphisms in a V. vinifera cultivar would indicate apparent genetic stability over the long period of cultivation of this cultivar. If V. vinifera cv Italia had an estimated 40 years of cultivation and showed no polymorphism at the Est, Mdh, Acp, Pod, Gtdh, Aat, and Alp genes examined, it may simply mean that the rate at which new alleles arise and became fixed in the population is slower than we predicted. Berry skin colour phenotype differences probably reflect polymorphism of genes controlling the pigment distribution in berry skin and not those associated with intermediary metabolism as tested. Tissue specific and variation in staining intensity of EST, MDH, POD, and GTDH isozymes is consistent with differential isozyme levels. Regulatory gene may be operative in determining the number of molecules of enzyme in a cell. The berry skin polymorphism in four V. vinifera cv also may be determined by regulatory genes. Analyses of change frequencies for berry skin colour indicate that berry skin colour polymorphism in Italia, Rubi, Benitaka, and Brasil grapes may be determined

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by at least four regulatory genes. A C gene that regulates the induction of all structural genes for the production of anthocyanin compounds (Boss et al., 1996) and permits colour expression in Rubi and Benitaka cultivars. In Italia cv the allele c prevents colour expression, whereas cc constitution shows an epistatic effect with regard to anthocyanin biosynthesis in grape berries. It seems that mutation of epistatic cc constitution is the responsible event, since it originates from Italia cv and exhibits a berry skin of a light rosy red colour. The occurrence of mutant sectors for berry skin colour in Italia cv was not reported during the 1991e2002 period. Producers of other regions have also registered absence of mutant sectors in Italia cv which might have originated Rubi and Benitaka cultivars. It may thus be possible that occurrence of mutant sectors in Italia cv starting Rubi and Benitaka cultivars related by Kishino and Mashima (1980) and Sousa (1996) have occurred only once. Light colour in berries of Rubi cv may be due to an uneven distribution of anthocyanin pigments in the berry skin. It appears that D gene controls the distribution and intensity of the pigment in the berry skin. The genotype dd dilutes the colour, while genotypes DD and Dd permit the full expression of skin colour. The occurrence of mutant sectors in Benitaka cv, which started the Brasil cv, has been detected in five vines from three Benitaka cv vineyards in Marialva during the 1995e2002 period. Data contrast observations for changes in berry skin colours in the Italia cv. There is approximately a 0.1% occurrence frequency of sectors with the black colour in the berry skin of Benitaka cv. Current authors have also detected 40 Benitaka cv and 30 Rubi cv vines from 10 vineyard sectors which exhibited the Italia cultivars’ green colour of the berry skin. This phenotypic reversion occurred either on a side branch or on the same bunch of the cluster (Fig. 4). Likewise, there were sectors in Brasil cv (200 vines from 6 vineyards) which exhibited the berry skin red colour so characteristic of Benitaka cultivars. Reversal to Italia phenotype in Benitaka (0.4%) and Rubi (0.27%) cultivars indicates recovery of the cc homozygous constitution in sectors (side branch or bunch) of Benitaka and Rubi Cc genotype. Since mitotic crossing-over may promote homozygotization of genes in the heterozygous condition, the possible occurrence of a recombination event at the Cc locus may be taken into account. Although reverse mutation may be an alternative explication, it seems unlikely since reversals are rare. Although somatic rearrangements are also considered rare events, the occurrence of somatic crossing-over in plants has been reported as being induced by chemical and physical agents (Arenaz and Vig, 1978; Vig, 1982). It has also been induced in in vitro tissue culture (Machado et al., 2000), and may be induced in V. vinifera cv by transposable genetic elements which can mediate the chromosomal rearrangements. Somatic rearrangements may also explain the origin of the Brasil cv from Benitaka cv (0.1%), as well the former’s reversal to the Benitaka phenotype in Brasil cv (28.57%). Somatic crossing-over may be occurring in genes encoding enzymes for the modification of anthocyanins in some sectors of Benitaka that constituted the Brasil cv. Martin and Gerats (1993) report that the fine adjustment in floral colour variety in Perilla frutescens var. crispa is determined by gene activity which encodes enzymes for anthocyanin modification, such as glycosilation, acylation, and methylation. A 5-GT gene (UDP-glucose: anthocyanin 5-O-glucosyltransferase) in

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Fig. 4. Reversal to Italia phenotype occurring in one bunch of the Benitaka cultivar (Cc genotype).

P. frutescens was specifically expressed in leaf of the red type, but not in those of the green one (Yamazaki et al., 1999, 2003). V. vinifera varieties usually produce different anthocyanin types and quantities; 17 anthocyanins were found in samples of V. vinifera cv Shiraz (Boss et al., 1996) in which the most abundant anthocyanin kinds were of three types. Predominant types may be determined by enzymeencoding gene activity for anthocyanin modifications. We may thus suggest that in Benitaka cv the genotype MM and Mm encoding enzymes that determine the anthocyanin type shows the rosy red colour in the berry skin of grapes. Somatic crossing-over in Mm genotype sectors may be producing mm constitution that induces an altered expression for anthocyanin modification and marks the dark colour (black or purplish red) of the Brasil cv’s berry skin. Reversal to the Benitaka cv phenotype in sectors of Brasil cv (28.57%) may be occurring through the suppressor allele (su) which cancels the expression of m allele and restores the corresponding M_ phenotype. Somatic crossing-over in a suppressor gene (Susu) of the Brasil cv sectors produces the susu constitution in side branches and berries. The mm/susu genotype will be thus a rosy red (M-like) phenotype. However, the frequency of reversion to Benitaka from Brasil cv is much higher than the other changes, thus the most plausible explanation is that insertion-sequence elements can appear in the middle of gene interrupting the coding sequence and inactivate the expression of M_ gene, or that Brasil cv is a periclinal chimera containing tissue in which the colour change has occurred (Ccmm) and no change in

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tissue (CcMm and CcMM ). Franks et al. (2002) have reported clonal differences that affect cultivar identification and pedigree analysis of grapevine of Pinot Meunier. Studies on plants and yeast indicate that somatic recombination events have been frequently associated with occurrence of induced mutations (Gorbunova et al., 2000; Baptista and Castro-Prado, 2002). Double-strand breaks of DNA may stimulate recombination during mitotic division. In callus tissue culture, the occurrence of somatic crossing-over has been suggested as one of the possible mechanisms inducing somaclonal variation (Machado et al., 2000). Morphological traits of somaclones generated in in vitro regenerated shoots reproduce the morphological traits of varieties and species present in natural populations of genus (Mangolin et al., 1997). Our data of isozyme analysis indicate the involvement of regulatory genes determining differential expression for constitutive genes in seven enzymatic systems as well as determining the berry skin colour polymorphism in grape cultivars of V. vinifera growing in the Marialva region. Italia, Rubi, Benitaka, and Brasil seem to be clones of the same cultivar. The berry skin colour polymorphism analyses suggest simply the occurrence of somatic crossing-over in naturally cultivated plants and a periclinal chimerism in Brasil cv. DNA fingerprint should be used to study the relationship existing among these four grape cultivars. References Almeida, J., Carpenter, R., Robbins, T.P., Martin, C., Cohen, E.S., 1989. Genetic interactions underlying flower color patterns in Antirrhinum majus. Genes Dev. 3, 1758e1767. Altube, H., Cabello, F., Ortiz, J.M., 1991. Characterization of grape varieties and rootstocks by isozymes from woody parts. Vitis 30, 203e212. Arenaz, P., Vig, B.K., 1978. Somatic crossing-over in Glycine max (L.) merrill: activation of dimethyl nitrosamine by plant seed and comparison with methyl nitrosourea in inducing somatic mosaicism. Mutat. Res. 52, 367e380. Baptista, F., Castro-Prado, M.A.A., 2002. uvsZ1 mutation shows epistatic relations with uvsD153 and uvsJ1 mutations without any involvement with checkpoint control in Aspergillu nidulans. Biol. Res. 35, 441e446. Beld, M.G.H.M., Martin, C., Huits, H., Stuije, A.R., Gerats, A.G.M., 1989. Flavonoid synthesis in Petunia: partial characterization of dihydroflavonoid 4-reductase genes. Plant Mol. Biol. 13, 491e502. Boss, P.K., Davis, C., Robinson, S., 1996. Analysis of the expression of anthocyanin pathway genes in developing Vitis vinifera L. cv Shiraz grape berries and the implications for pathway regulation. Plant Physiol. 111, 1059e1066. Camargo, H.A., 1994. Uvas do Brasil. Empresa Brasileira de Pesquisa Agropecua´ria, Centro Nacional de Pesquisa de Uva e Vinho. Embrapa-SPI, Brası´ lia, 90 p. Douce, R., Neuburger, M., 1990. Metabolic exchange between the mitochondrion and the cytosol. In: Dennis, D.T., Turpin, D.H. (Eds.), Plant Physiology Biochemistry and Molecular Biology. Longman Scientific & Technical, New York, pp. 173e190. Eiras-Dias, J.E.J., Sousa, B., Cabral, F., Carvalho, I., 1989. Isoenzymatic characterization of Portuguese vine varieties of Vitis vinifera L. Riv. Vitic. Enol. 1, 23e26. Franks, T., Botta, R., Thomas, M.R., Franks, J., 2002. Chimerism in grapevines: implications for cultivar identity, ancestry and genetic improvement. Theor. Appl. Genet. 104, 192e199. Gonc¸alves, J.A., 1995. Parana´ descobre nova variedade de uva. Folha de Sa˜o Paulo, Agrofolha, Sa˜o Paulo, 12 dezembro, pp. 1. Gorbunova, V., Avivi-Ragolski, N., Shalev, G., Kovalchuk, I., Abbo, S., Hohn, B., Levy, A.A., 2000. A new hyperrecombinagenic mutant of Nicotiana tabacum. Plant J. 24, 601e611.

702

S.A. de Oliveira Collet et al. / Biochemical Systematics and Ecology 33 (2005) 691e703

Griffiths, A.J.F., Miller, J.H., Susuki, D., Lewontin, R.C., Gelbart, W.M., 1996. Gene mutation. In: An Introduction to Genetic Analysis, sixth ed. W.H. Freeman and Company, New York, pp. 181e210. Jorge, I.C., Mangolin, C.A., Machado, M.F.P.S., 1997. Malate dehydrogenase isozymes (MDH; EC 1.1.1.37) in long-term callus culture of Cereus peruvianus (Cactaceae) exposed to sugar and temperature stress. Biochem. Genet. 35, 155e159. Kishino, A.Y., Mashima, M., 1980. Uva: Vitis vinifera L. In: Manual Agropecua´rio do Parana´. IAPAR, Londrina, pp. 138e177. Ludwing, S.R., Wessler, S.R., 1990. Maize R gene family: tissue-specific helix-loop-helix proteins. Cell 62, 849e851. Machado, M.F.P.S., Mangolin, C.A., Oliveira-Collet, S.A., 2000. Somatic crossing-over can induce isozyme variation in somaclones of Cereus peruvianus Mill. (Cactaceae). Haseltonia 7, 77e80. Machado, M.F.P.S., Prioli, A.J., Mangolin, C.A., 1993. Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes in tissue and callus tissue of Cereus peruvianus (Cactaceae). Biochem. Genet. 31, 167e171. Mangolin, C.A., Prioli, A.J., Machado, M.F.P.S., 1997. Isozyme variability in plants regenerated from calli tissue of Cereus peruvianus (Cactaceae). Biochem. Genet. 35, 189e204. Martin, C., Gerats, T., 1993. The control of flower coloration. In: Jordan, B.R. (Ed.), The Molecular Biology of Flowering. CAB International, Wallingford, CT, pp. 219e255. Martin, C., Prescott, A., Mackay, S., Bartlett, J., Vrijlandt, E., 1991. The control of anthocyanin biosynthesis in flowers of Antirrhinum majus. Plant J. 1, 37e49. Newton, K.J., 1983. Genetics of mitochondrial isozymes. In: Tanksley, S.D., Orton, T.J. (Eds.), Isozymes in Plant Genetics and Breeding, Part A. Elsevier, Amsterdan, pp. 157e174. Or, E., Baybik, J., Sadka, A., Zaks, Y., 2000. Isolation of mitochondrial malate dehydrogenase and phosphoenolpyruvate carboxylase cDNA clones from grape berries and analysis of their expression pattern throughout berry development. J. Plant Physiol. 157, 527e534. Orasmo, G.R., Machado, M.F.P.S., 2003. Isozyme diversity in RB (Republic of Brazil) sugarcane varieties (Saccharum spp). Acta Sci. 25, 213e219. Pasteur, N., Pasyeur, G., Bonhomme, F., Catalan, J., Britton-Davidian, J., 1988. Genetic interpretation of gels. In: Practical Isozyme Genetics. Johhn Wiley & Sons, Ellis Horwood, pp. 31e48. Quattrocchio, F., Wing, J.F., Leppen, H.T.C., Mol, J.N.M., Koes, R.E., 1993. Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets of target genes. Plant Cell 5, 1497e1512. Resende, A.G., Vidigal-Filho, P.S., Machado, M.F.P.S., 2000. Isozyme diversity in cassava cultivars (Manihot esculenta Crantz). Biochem. Genet. 38, 203e216. Royo, J.B., Cabello, F., Miranda, S., Gogorcena, Y., Gonzalez, J., Moreno, S., Itoiz, R., Ortiz, J.M., 1997. The use of isozymes in characterization of grapevines (Vitis vinifera, L.). Influence of the environment and time of sampling. Sci. Hortic. 69, 145e155. Sanches-Escribano, E., Ortiz, J.M., Cenis, J.L., 1998. Identification of table grape cultivars (Vitis vinifera L.) by the isozymes from the woody stems. Genet. Resour. Crop Evol. 42, 173e179. Schaefer, H., 1971. Disk-elktrophorestischer nachiveis der polyphenoloxydasen aud rebenbla¨ttern. Vitis 10, 31e32. Sousa, J.S.I., 1996. Uvas para o Brasil (Rev. Aum.), second ed. FEALQ, Piracicaba, 791 p. Subden, R.E., Carey, K., Meiering, A.G., Krizus, A., 1992. Genetic stability in the Vitis vinifera L. cv. ‘Chardonnay’ wild accession of Vitis riparia Michx and hybrid cv. ‘Seyval Blanc’. Can. J. Plant Sci. 72, 875e882. Taureilles, C., Robin, J.P., Flanzy, C., 1992. Grape (Vitis vinifera L.) malate dehydrogenase: partial purification and isoelectric heterogeneity. Sci. Aliments 12, 649e663. Tedesco, G., Villa, P., Valentin, L., Sala, E., Scienza, A., 1997. A chemotaxonomic investigation on Vitis vinifera L. III: Characterization of Vitis biotypes via root apex proteins. Vitis 36, 85e89. Vig, B.K., 1982. Soybean (Glycine max [L.] merrill ) as a short-term assay for study of environmental mutagens. A report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat. Res. 99, 339e347. Vouillamoz, J., Maigre, D., Meredith, C.P., 2003. Microsatellite analysis of ancient alpine grape cultivars: pedigree reconstruction of Vitis vinifera L. ‘Cornalin du Valais’. Theor. Appl. Genet. 107, 448e454.

S.A. de Oliveira Collet et al. / Biochemical Systematics and Ecology 33 (2005) 691e703

703

Weeden, N.F., Reisch, B.I., Martens, M.H.E., 1988. Genetic analysis of isozyme polymorphism in grape. J. Am. Soc. Hortic. Sci. 113, 765e769. Yamazaki, M., Gong, Z., Fukuchi-Mizutani, M., Fukui, Y., Tanaka, Y., Kuusumi, T., Saito, K., 1999. Molecular cloning and biochemical characterization of a novel anthocyanin 5-O-glucosyltransferase by mRNA differential display for plant forms regarding anthocyanin. J. Biol. Chem. 274, 7405e7411. Yamazaki, M., Nakajima, J., Yamanashi, M., Sugiyama, M., Makita, Y., Springob, K., Awazuhara, M., Saito, K., 2003. Metabolomics and differential gene expression in anthocyanin chemo-varietal forms of Perilla frutescens. Phytochemistry 62, 987e995.