DIFFERENCES IN TOMATO SEED PROTEIN PROFILES OBTAINED BY SDS-PAGE ANALYSIS

The protein profiles of tomato seeds from sub-species ( subsp. cultum Brezh., subsp. subspontaneum Brezh. and subsp. spontaneum Brezh.) were analyzed using SDS-PAGE technique. Electrophoreograms and denzitograms of total, soluble and non-soluble proteins of 31 different samples have showed quantitative and qualitative differences. Qualitative differences in electrophoregrams of total seed proteins refer to protein fragments in zone A (114 kDa, 83 kDa and 65 kDa) and protein fragment in zone C (17 kDa). Qualitative differences in electrophoregrams of soluble seed proteins refer to protein fragment in zone A (94 kDa). Qualitative differences in electrophoregrams of nonsoluble seed proteins refer to protein fragments with molecular weights of: 210 kDa, 85 kDa, 67 kDa and 26 kDa.


Introduction
Endosperm seed protein analysis constitutes a valid and/or improved approach to cultivar identification which is commonly based on morphological traits recorded in the field.Seed storage proteins analyses results are very useful for differentiating and characterizing the studied ecotypes ( Mennella et al., 2001).
Specific proteins and enzymes as markers have application in selection of parental material for hybridization, cultivar purity testing, explanation of phylog-eny and taxonomy relations among different genus and species, F 1 hybrid purity testing etc. ( Markova et al., 2003).Analyses of seed storage proteins in many horticultural species allowed the differentiation among species, cultivars and androgenetic lines ( Kononkov et al ., 1987;Smith and Smith, 1992;Dinelli and Bonetti, 1992;Cooke, 1995;Mennella et al., 1995Mennella et al., , 1996Mennella et al., , 1998Mennella et al., , 1999)).
For variety identification and genetic purity control of self-pollinated and open-pollinated species, storage and functional proteins as direct gene products could be used.These genetic markers are widely used as tools for the identification and estimation of the quantitative traits in plant resources, resistant to disease and environmental stress conditions, and other desirable agronomic traits ( Zlokolica et al., 1997).
The electrophoretic protein profiles and their high stability and independence of the ecological conditions could be used as genotype markers ( Sullivan and Freytag , 1986;Lioi, 1991;Stoyanova et al., 1992).The information that protein electrophoreograms gives us, can assist to the process of valid selection of starting hybridization material ( Konarev, 1983).
The purpose of this study was to detect differences in seed protein profiles of assayed tomatoes and possibility for their use in identification of different tomato lines, hybrids, populations and varieties.
Protein extraction: Tomato seed proteins were extracted using the method described by S. Doonan (1996 and modified by G.D. Efremov (1998).Exact quantity of each seed was homogenized in electric mixer and proteins were extracted using two procedures.
Soluble and non-soluble proteins were extracted in one fraction using procedure A. Powdered seed was immersed in buffer A (0,0625 M TRIS-HCl (pH 6.8), 2% (W/V) SDS, 5% (V/V) -mercaptoethanol, 10% (W/V) glycerol and 0,002% (W/V) bromphenol blue) in proportion 1:2,5.The seed sample was homogenized by vigorous vortexing, kept at room temperature for 2 hours, and denaturized at 95 o C in water bath for 2 minutes.After this treatment the sample was centrifuged at 10 000 -20 000 rpm, and the supernatant was used for electrophoretic analysis.
Procedure B was performed in order to get the soluble and non-soluble proteins in two separate fractions.For that purpose powdered seed was immersed with buffer B which contained 0,1M TRIS-HCl (pH 8.0), 0,01 M MgCl 2 , 18% (W/V) sucrose and 40 mM -mercaptoethanol in proportion 1:3.In the first step after the homogenization, procedure A was performed and the soluble proteins were extracted in supernatant.In the second step non-soluble proteins were extracted by rinsing of the sediment with the solution which contained 150l 2% (W/V) SDS, 50l 6% (W/V) sucrose and 40 mM -mercaptoethanol.After the homogenization by vortexing the procedure A was repeated.
The gel was stained using 1% Commassie Blue G-250 in buffer that consisted: 30% methanol and 7% acetic acid.The gel was unstained by the same buffer.Obtained electrophoreograms were analyzed using PHOTO-CAPT SOFTWARE program and for each protein profile was obtained denzitogram.

Results and Discussion
Tomato seed proteins separated by SDS-PAGE were divided in three zones: A, B and C from cathode to the anode.Zone C contains protein fragments with smaller molecular weight, zone B contains proteins fragments with medium molecular weight and zone A contains protein fragments with the biggest molecular weight ( Fig. 1  Analyses of SDS-PAGE electrophoregrams and denzitograms showed quantitative and qualitative differences between soluble seed protein profiles in the three zones.Qualitative differences refer to protein fragment with molecular weight of 94 kDa in zone A. The protein fragment was not identified only in Lycopersicon esculentum Mill.subsp.cultum Brezh.hybrid 447 (Fig 3).In the zone C appeared protein fragments with following molecular weights: 28 kDa, 25 kDa, 20 kDa, 18 kDa, 16 kDa and 14 kDa.Protein differences also were found in number of fragments in zone C. In Lycopersicon esculentum Mill.subsp.cultum Brezh.(L-63-97,L-5/91, hybrid 127, hybrid 261, L-26/97, L-6/97, hybrid 14, hybrid 158, L-223/92, L-136/98, hybrid 230 and hybrid 670) 4, 5 or 6 protein fragments were noticed, that are more compared to other analyzed tomatoes.
SDS-PAGE electrophoreograms and denzitograms of non-soluble proteins showed quantitative differences in intensity of some protein fragments in the three zones of protein profiles.The protein fragment with the molecular weight of 210 kDa was found in Lycopersicon esculentum Mill.subsp.cultum Brezh.(hybrid 677, hybrid 127, hybrid 261, L-26/97, L-6/97, hybrid 14, hybrid 158, L-223/92, L-136/98 and hybrid 230) (Fig. 4).85  Differences between protein profiles in different tomatoes mainly were quantitative.Qualitative differences in SDS-PAGE electrophoregrams of total seed proteins refer to protein fragments with molecular weights of 114 kDa, 83 kDa, 65 kDa and 17 kDa.Qualitative differences in SDS-PAGE electrophoregrams of soluble seed proteins refer to protein fragment in zone A with molecular weight of 94 kDa.Qualitative differences in SDS-PAGE electrophoregrams of non-soluble seed proteins refer to protein fragments with molecular weights of: 210 kDa, 85 kDa, 67 kDa and 26 kDa.Protein fragments that were detected as qualitative differences in SDS-PAGE electrophoreograms of assayed tomatoes are presented in Table 2.A weak polymorphism in SDS-PAGE banding patterns of L. esculentum Mill.ecotypes was detected and also by Mennella et al. (2001).

Conclusion
In this study, detected differences in seed protein profiles of analyzed tomatoes were not enough to use them in identification of different tomato lines, hybrids, populations and varieties.It is obvious that morphological and biochemical methods are not alternative methods, but they are complementary between each other.

Table 2 .
Detected qualitative differences in assayed tomatoes