Abstract
Two isoforms of ascorbate peroxidase, namely, APX 1 and APX 2 with subunit molecular weight of 28.5 and 34 kDa, respectively, were purified from leaves of 15-day grown rice ( Oryza sativa L.) plants. The molecular and enzymatic properties of the two isoforms differed significantly but both belonged to the class of ascorbate specific heme peroxidases and were inhibited by thiol reagents. The two isozymes were separated from each other at the DEAE-Sephacel chromatography step of purification and were named APX 1 and APX 2 in the order of elution in the chromatography. APX 2 accounted for 74% of the total activity. The Km values of APX 1 and APX 2 for ascorbate were 400 and 200 μM and those for hydrogen peroxide were 33 and 76 μM, respectively. Both isoforms had very high preference for ascorbate as electron donor. pH optima for APX 1 and APX 2 were 6.5 and 7.0, respectively. Activity of both isoforms was completely lost by heat treatment at 80 °C for 20 min or in presence of 150 mM NaCl in the assay medium. A 40% polyethylene glycol (PEG-6000) in the assay medium led to about 50% inhibition in the activity of APX 1 and 65% inhibition of APX 2 with an increase in enzyme Km values. Incorporation of 1 M proline, glycine betaine or sucrose in presence of PEG in the assay medium led to a pronounced restoration in enzyme activity with decrease in Km values. Glycine betaine appeared to be an effective protectant for both isoforms under in vitro water stressed conditions. APX 2 appeared to be more sensitive to the metals Ni, Al and As when compared to APX 1. When rice seedlings were raised in sand cultures in nutrient solution containing 1 mM Al 2(SO 4) 3, during a 5–20 day growth period about 0.5–1.3 times higher activity of APX was observed compared to the activity in controls. Similarly, rice seedlings subjected to polyethylene glycol induced water deficit for 24 h showed elevated APX activity compared to unstressed seedlings. Ascorbate peroxidase in rice seedlings appears to have an important role in detoxification of H 2O 2 under abiotic stressful conditions.
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