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Scientia Agricola

On-line version ISSN 1678-992X

Sci. agric. vol. 54 n. 3 Piracicaba Sep./Dec. 1997 



1Instituto de Biociências/UNESP, CEP: 18618-000 - Botucatu, SP.
2Centro de Energia Nuclear na Agricultura/USP, CEP: 13416-000 - Piracicaba, SP.



ABSTRACT: One of the uses of the technique of tissue culture for plant breeding is the identification of cell lines tolerant to salt stress.In order to study the biochemical mechanisms involved in the genetic expression to salt tolerance, callus from embryo axis of four bean cultivars (cv. IAC-carioca; cv. IAPAR-14; cv. JALO-EEP558; CV. BAT-93) were grown in Murashige & Skoog (1962) medium, supplemented with NaCl in the concentrations of 0, 20, 40, 60 and 80 mM. After 14 days callus were harvested and analyzed according to their isoenzymatic patterns and peroxidase activities. BAT and IAPAR cultivars showed two common activity zones in the anodic region, with only one specific enzymatic band to each one (the two fastest migration band); it is possible that the two middle anodic zones detected are products of the same enzymatic locus but from different alleles with different eletrophoretic mobilities. Cv. JALO showed two anodic activities in common with cvs IAC and IAPAR with an exclusive anodic zone of slower migration which showed the most intense activity of all cultivars analyzed. This cv. still showed a dimeric heterozygotic catodic zone in all treated samples. Probably this is the same zone which occurs in homozygosis with fixation of the slower allele for all cvs BAT and IAPAR submitted to all treatments. Cv. IAC showed two anodic bands in common with Cv. IAPAR and cv. JALO. It still showed a faster anodic band in common with cv. IAPAR and an exclusive anodic band of slower migration. It is interesting to say that for this cv. IAC resulting from cultivation in NaCl 20 mM did not show activity in the three slower anodic zones. Cv. IAC showed only one dimeric heterozygotic catodic zone in all treatments. This zone is probably composed by two different alleles from the same locus detected in cv. JALO. Samples from cv. IAC treated with 40 and 60 mM showed a more intense enzymatic activity in the catodic zone. Analyses of the peroxidase activity in the crude extract of the different bean cultivars analysed showed different reations to salt concentration in the cultivation procedures as well as a high increasing of peroxidase activity in cv. IAC and JALO.
Key Words:
Phaseolus vulgaris, peroxidases, polymorphism, salt stress



RESUMO: Uma das utilizações da técnica de cultura de tecidos para o melhoramento vegetal é a identificação de linhas de células que apresentem tolerância ao estresse salino. Para se estudar os mecanismos bioquímicos envolvidos na expressão genética da tolerância a salinidade, calos oriundos de eixos embrionários de quatro cultivares de feijão (Phaseolus vulgaris L.; cultivares IAC - carioca, IAPAR 14, JALO-EEP 558, BAT - 93), foram cultivados em meio sólido Murashige & Skoog (1962), suplementado com NaCl nas concentrações de 0, 20, 40, 60 e 80 mM. Após 14 dias de incubação, os calos foram coletados e analisados quanto aos padrões isoenzimáticos e de atividade das peroxidases. Os cultivares BAT e IAPAR apresentaram duas zonas de atividade em comum na região anódica e apenas uma zona enzimática específica a cada um deles (migração mais rápida).Possivelmente as duas zonas anódicas intermediárias sejam produtos do mesmo loco enzimático, porém com alelos diferentes, consequentemente diferentes mobilidades eletroforéticas. O cv. JALO apresentou duas zonas anódicas de atividade em comum com os cultivares IAC e IAPAR com uma zona anódica exclusiva de migração mais lenta, a qual apresentou atividade mais intensa de todos os cultivares analisados. Este cultivar revelou ainda uma zona catódica provavelmente dimérica e heterozigota nos indivíduos de todos os tratamentos aplicados. Provavelmente, esta é a mesma zona que ocorre em homozigose com fixação do alelo lento para os indivíduos de todos os tratamentos efetuados nos cultivares BAT e IAPAR. O cv. IAC apresentou duas bandas anódicas em comum com os cv. IAPAR e JALO. Apresentou também a banda anódica mais rápida em comum com o cv. IAPAR e uma banda anódica exclusiva de migração mais lenta. Curiosamente, os indivíduos deste cv. mantidos em meio suplementado com 20 mM de NaCl não apresentaram atividade nas três zonas anódicas mais lentas. Ocorreu no cv. IAC uma única zona de atividade catódica, dimérica e heterozigota para os indivíduos provenientes de todos os tratamentos, composta provavelmente de dois alelos diferentes da zona correspondente ao cv. JALO. Amostras provenientes dos tratamentos 40 e 60 mM de NaCl, desta zona catódica, apresentaram maior atividade enzimática. A análise da atividade da peroxidase no extrato bruto, revelou que os cultivares responderam diferentemente ao aumento da concentração salina no meio de cultura, com aumento pronunciado dessa atividade nos cultivares IAC e JALO.
Phaseolus vulgaris, peroxidases, polimorfismo, estresse salino




Isoenzymatic patterns of several important enzymes can show variable responses to stressing factors.

Stevens et al. (1978), when studying peroxidase (E.C. activity as a selective parameter for tolerance to salt stress in Brassica, have not found a positive correlation between growth and change in enzyme activity.

Sahu & Mishra (1987) reported changes in enzymatic activity of peroxidase during senescence of rice leaves when submitted to salt stress. They observed that NaCl increased peroxidase activity which could be related to regulation of membrane permeability, cell wall formation and oxidation of accumulated substances due to salt stress.

Peroxidases are enzymes related to polymer synthesis in cell wall (Bowles, 1990), as well as with prevention of oxidation of membrane lipids (Kalir et al., 1984). According to Gaspar et al. (1985) an increase in peroxidase activity in cultivars sensitive to salt could be responsible for the ability of such cultivars to adapt to external stimulus.

Peroxidase isoenzyme characteristics in situ and in vitro were compared in 4 rice cultivars with different degrees of salt tolerance by Mittal & Dubey (1991). After 96 h of NaCl treatment under low concentration (90 mM), it was observed that more tolerant cultivars showed similar isoenzymatic pattern (5 bands). The highest the salt concentration in the medium (148 mM) the lowest the intensity in the first three bands. The present study aimed to observe the possible changes in isoenzymatic patterns and peroxidase activity in callus of 4 bean cultivars (Phaseolus vulgaris, L. IAC-carioca, IAPAR-14, JALO-EEP 558 and BAT-93 cultivars) under salt stress, after cultivation in semi-solid medium supplemented with NaCl in concentrations of 0; 20; 40; 60 and 80 mM.



Plant Material: Bean seeds (Phaseolus vulgaris, L.) of the cultivar IAC carioca, IAPAR-14, JALO-EEP 558 and BAT-93 were used.

Growth Conditions: Seeds were washed and disinfected in 20% commercial sodium hypochlorite solution (v/v; 1% of active chlorine) for 5 minutes. After rinsing seeds were left for 18 hours in distilled water for imbibition. Embryo axis were disinfected in the same way and inoculated in MS medium (Murashige & Skoog, 1962) and placed in a growth room at 25ºC and 2000 lux, with a photoperiod of 16/8 hours for callus induction.

Treatments: After the third subcultivation, calli were transferred to MS medium supplemented with 0; 20; 40; 60 and 80 mM NaCl and maintained under controlled conditions for 14 days. Three replicates for each treatment were used.

Extraction and isoenzyme electrophoresis: Fresh calli were collected, weighted and ground in Tris-HCl 0.1 M, pH 7,0 (Galgaro, 1991). After centrifuging (6,048 g) for 10 minutes, at 5°C, the supernatant was collected and frozen. Samples were applied with Wahtman # 3 filter, on penetrose gel 12%, using 0.135 M Tris-citrate, pH 7.0 for tubes and diluted (1:14) for gels (Soltis & Soltis, 1989). Electrophoresis was conducted in a refrigerator (4°C) at 20 mA for 40 minutes initially, and increasing to 50 mA until indicator (bromophenol blue) reached the anode.

Staining: Peroxidase isoenzymes were detected with diluted orthodianisidine in 50 mL of acetate 0.2 M, pH 5.0, then adding 2 mL of 3% hydrogen peroxide.

Peroxidase specific activity: Callus samples (1g FW) were ground in 5 mL of 0.2M, phosphate buffer 0.2M, pH 6.7. Afterwards the extract was centrifuged for 10 minutes at 4°C at 12.100 g. The supernatant was collected and and frozen.

For enzyme activity, 0,5 mL of H2O2 solution (35% diluted in 0.2M phosphate buffer pH 6.7) plus 0.5 mL of 4-aminoantipyrine/phenol solution were added to 200 ml of crude extract. After incubation for 5 minutes at 30°C the reaction was stopped with 2 mL of ethanol. Optic density was obtained at 505 nm in a spectrophotometer (Allain et al., 1974). The specific activity of peroxidase were expressed in mmol H2O2.min. mg protein-1. In all enzyme preparations, total protein was estimated by the method of Bradford (1976) using bovine serum albumin (BSA) as standard.



Peroxidase electrophoretic analysis: Four activity zones were detected in the anodic region of the gel for all cultivars except for cv. JALO, which did not show zone # 4, but showed a high peroxidase activity for all the other three zones (Figure 1).

Different electrophoretic patterns among cultivars with were observed this enzimatic system. When different NaCl concentrations were utilized, only cv. IAC showed an absence of bands in zones 1, 2 and 3. The same cv. presented also a more intense intermediary catodic bands for a samples treated with NaCl 40 and 60 mM (Figure 1).

Although cv. IAC showed three exclusive patterns among samples, this variation seems to have no correlation with the salt levels in the medium.


54n3a3f1.GIF (2733 bytes)

Figure 1 - Peroxidase eletroforetic paterns in callus of four beans cultivars cultivated under salt stress. 1,2,3,4 and 5 represent the salt treatments (0, 20, 40, 60, 80 x 10-3 mol. l-1).


In this enzimatic system, the only catodic zone detected in the gel (Figure 1) showed inter-cultivars variation in relation to the number of detected bands for each cultivars as well as electrophoretic mobility of each one.

One slow catodic band shared by cultivars BAT and IAPAR was not detected in cv. JALO and IAC. These two last cultivars showed 3 bands with higher mobility than the one shared by BAT and IAPAR, suggesting the presence of monomeric and dimeric forms for this enzyme as described by Garcia et al., (1982), and Brewbaker et al. (1985) working with rye and maize, respectively.

It was not possible to observe the relationship between the salt levels in the medium and banding patterns. The same results were observed by Stevens et al. (1978) studying salt stress tolerance in Brassica.

Cahwla (1988) studyed isoenzymatic changes for peroxidase, esterases and acid phosphatase in morphogenesis of barley and wheat calli. The author observed that such enzymes could not be used as markers to distinguish morphogenetic calli from those not morphogenetic, because the results could not be extended to another organs in intacts plants.

Liu & Li (1991) studying the effect of NaCl in peroxidase isoenzyme patterns of root and regenerated callus leaves of tomato, observed the presence of identical isoenzymatic bands. They demonstrated that NaCl did not influence the peroxidase patterns.

Zhou et al. (1992), when analyzing the relationship between different hormonal concentrations and electrophoretic patterns of peroxidase during somatic embryogenesis in lettuce cotyledons, observed that embrioneus callus bands were bigger and more intense than those of non-embriogenic callus. Therefore, it was possible to suggest that quality modifications in isoperoxidase band patterns were more related to the beginning of organogenesis and tissue differentiation, than to environmental factors, as salt stress and hormonal balance. Although these factors can influence callus development, they are not directly related to new peroxidase forms and their intensity.

Peroxidase Enzymatic Activity in Crude Extract: The cultivars had different reactions to the increasing salt concentration in the medium. IAC and JALO cultivars showed an increase in peroxidase activity above 20 mM of NaCl while cv. IAPAR and BAT did not show significant raise of enzymatic activity, except at the concentration of 20 mM of NaCl (Figure 2).


54n3a3f2.GIF (3808 bytes)

Figure 2 - Peroxidase activity in callus of four beans cultivars, submited to salt stress; prot. Vertical bars indicates ± SE.


Injuries in plants caused by salt stress were classified by Levitt (1980) in primary and secondary. Primary stress causes physiological harm in membrane level. Indirectly salt can interfere in protein metabolism causing enzymatic activity changes and other metabolic disturbances.

Rakova et al. (1969), Siegel (1993) and Zhang & Kirkham (1994) stated that peroxidase, as well as catalases and amilases, can react to salt with an increase or decrease in their activities due to the concentration in medium. Strogonov (1980) has also observed an increase in peroxidase activity in rice leaves, injured by NaCl. This enzyme has an important role in oxidation of accumulated substances, due to stress caused by salt.

Production of great quantities of H2O2 as a response to hydric stress, can lead to peroxidase liberation at membrane level, in which enzymes are usually associated, as in spinach cloroplast (Groden & Beck, 1979). Hydric stress can also induce, in natural substracts, accumulation of peroxidase such as glutatione, ascorbic acid and phenolic compounds which are oxydized by aerobic cells by peroxidases (Winston, 1990).

As a consequence, cells have begun a degradation process of proteins by hydrolysis. An increase in peroxidase activity in cv. IAC and JALO could have occurred due to a disorganization in permeability of membranes and metabolite oxidation, as a reaction to NaCl. It could also be concluded that cv. IAPAR and BAT showed a higher potential to salt tolerance when comparing to cv. IAC and JALO by considering different responses to peroxidase activity.


This research was supported by FUNDUNESP # 122/94.



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Recebido para publicação em 22.10.96
Aceito para publicação em 25.04.97

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