Aluminium induced esterase activity and isozyme pattern in barley root tip

Abstract

Changes in the activity of esterase as well as changes in the viability of root cells and some growth parameters were analysed during cultivation of barley seedlings in the artificial substrate under Al stress conditions. Aluminium-induced elevated esterase activity correlated with Al uptake, root growth inhibition and increased Evans blue uptake in the barley root tips. Analysis of isozyme pa�ern of esterase revealed one anodic and one cathodic esterase isozyme induced by Al-treatment. The possible role of elevated esterase activity during Al stress is discussed.Keywords: aluminium uptake; cell death; esterase activity; isoesterases 221 Aluminium uptakeHematoxylin staining was used for the determination of Al uptake (Ownby 1993). Freshly harvested roots, washed in distilled water for 15 min were stained with 0.2% hematoxylin and 0.02% KIO 3 solution for 15 min at room temperature. After washing with distilled water for 15 min 10 root tips (0.5 cm) were excised and soaked in 200 µl of 1M HCl for 1 h. Optical density of the released stain was measured at 490 nm. Determination of cell deathThe loss of plasma membrane integrity was evaluated by spectrophotometric assay of Evans blue staining (Baker and Mock 1994). Roots were stained in 0.25% (v/v) aqueous solution of Evans blue for 15 min at room temperature. The stained roots were washed three times with distilled water, for 10 min each. Root tips (5 mm) were excised and soaked in N,N-dimethylformamide for 1 h at RT. The optical density was measured spectrophotometrically at 600 nm. Protein extraction and sample preparationRoot tips (1 cm) were ground to a fine powder in a cold mortar in liquid nitrogen and the resulting powder was rehomogenised in 100mM Tris/HCl buffer, pH 8.0 with homogenisator (Heidolph DIAX 900). After filtration the homogenate was centrifuged at 1500 g for 5 min, then at 12 000 g for 15 min and finally at 100 000 g for 30 min (Beckman L8-M). The resulting supernatant (soluble fraction) was used for analysis after concentrating and passing through Sephadex G-25 using 10mM Tris/HCl buffer, pH 8.0. Proteins were fractionated using anion exchange column (UNO Q, Bio-Rad) equilibrated with 10mM Tris/HCl buffer, pH 8.0. The adsorbed proteins were eluted with linear gradient of 0-1.0M NaCl in the same buffer. Proteins were quantified with Bovine Serum Albumin as a standard by the method of Bradford (Bradford 1976). Enzyme assaysEnzyme activities were determined photometrically using microplate reader (SLT-Laborinstruments, Austria). Specific enzyme activities were expressed as OD/µg protein (OD -optical density). Changes in enzyme activities were expressed as a percentage of control. Assay for non-specific esterase (EC 3.1.1.3) contained 100 µl of 100mM Tris/HCl buffer, pH 7.2; 50 µl 4-nitrophenylacetate (2 mg/ml solubilized in 20% acetone) and sampled according to Ward and Bamforth (2002). Activity was measured at 405 nm against the control reaction without sample. Gel electrophoresis and activity stainingThe basic proteins were separated u...

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