Quantificação dos níveis endógenos de auxinas e da actividade enzimática das polifenoloxidases em oliveira (Olea europaea L.)

Quantification of endogenous auxin levels and polyphenoloxidase enzymatic activity in olive (Olea europaea L.)

 

C. Serra¹, R. Antunes¹, H. Hegewald², C. Costa³, A. P. Pinto^1,4 & A. Peixe⁴

 

RESUMO

A actividade enzimática de polifenoloxidases foi avaliada em folhas e na zona apical, média e basal de ramos de duas cultivares de oliveira (Olea europaea L.), comuns no Alentejo (‘Galega vulgar’ e ‘Cobrançosa’), mostrando que a actividade enzimática nas folhas foi muito superior à encontrada em tecidos de ramos do ano. Maior actividade enzimática foi também detectada na variedade ‘Cobrançosa’ versus ‘Galega vulgar’. As condições óptimas para a determinação da actividade enzimática foram: pH= 5.5 e T= 40 ºC, com 20 mM de 4-metilcatecol em tampão acetato. Nestas condições o KM determinado foi: 2,60 e 3,48 mM com o método de Michaelis-Menten e Lineweaver-Burk, respectivamente.

A melhor recuperação das auxinas AIA (ácido indol-3-acético) e AIB (ácido indolbutírico) em material vegetal foi conseguida através da extracção das amostras com acetona. A separação, identificação e quantificação do AIA e AIB em padrões, material vegetal dopado (tecidos de oliveira dopados com uma concentração conhecida de padrão) e não dopado, foi efectuada por técnicas cromatográficas (HPLC-DAD e LCMS), mostrando os resultados taxas de recuperação superiores a 40% para o AIA e 60% para o AIB.

 

ABSTRACT

The poliphenoloxidase enzymatic activity was evaluated in two olive cultivars (Olea europaea L.) widespread in Alentejo (‘Galega vulgar and ‘Cobrançosa). Leaves and apical, medium and basal regions of the year stems were used as sample material.

When compared with the different regions of the year stem, the results have shown that enzymatic activity was significantly higher in the leaves of both cultivars. Between cultivars, it was observed that ‘Cobrançosa’ presented higher enzymatic activity than ‘Galega vulgar’. The pH at 5.5 and 40 ºC temperature, using 20 mM of 4methylcatecol in acetate buffer were the optimized conditions for the enzymatic analysis. Under these conditions, the measured KM was 2,60 and 3,48 mM, using the Michaelis-Menten and the Lineweaver-Burk methods respectively.

The auxins IBA and IAA were removed from the plant material by an extraction with acetone. The separation, identification and quantification of IBA and IAA using vegetal plant material treated and non treated with exogenous auxins, was done by chromatographic techniques (HPLC-DAD and LC-MS). The sample preparation recoveries were higher than 40% for IAA, and higher than 60% for IBA.

 

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¹ Departamento de Química, Universidade de Évora, Rua Romão Ramalho nº 59, 7000 Évora; e-mail: app@uevora.pt; ²Lacrome Bairro do Bacelo, Rua César Batista, 6 D. 7000-715 Évora; ³Centro de Química de Évora, Universidade de Évora; ⁴Instituto de Ciências Agrárias Mediterrâneas (ICAM), Universidade de Évora