Changes in the Activity of Some Hydrolases, Peroxidase, and Catalase in the Rice Seed during Germination

Abstract

A study was made of the changes in activity of enzymes involved in the breakdown of stored phytin, lipid, and hemicellulose in the aleurone layer of rice seed (Oryza sativa L., variety 1R8) during the 1st week of germination in the light. Enzyme assays were made on crude extracts from degermed seed, and activities were expressed on a per seed basis. Phytase activity increased within the 1st day of germination. The increase in activity of most other enzymes-phosphomonoester. ase, phosphodiesterase, esterase, lipase, peroxidase, catalase, 5-glucosidase, and a-and~-galactosidase-closely followed the increase in protein content. Their peak activities occurred by the 5th to the 7th day. Some enzymes, such as 5-1,3-glucanase and a-amylase, continued to increase in activity after the 7th day. Phytase, L-1 ,3-glucanase, and a-amylase followed a similar sequence of production in embryoless seed halves incubated in 0.12 AM gibberellin A3, but the production of lipase was delayed.The aleurone layer of cereals is the site of production of hydrolases during seed germination and during incubation of embryoless seed halves or isolated aleurone layers in GA3 (2, 6-8, 19, 21, 24). Many hydrolases have been studied, including a-amylase (2, 19), protease (6,19), phytase (10, 15), phosphatase (24), lipase (26), and RNase (2,19). Phytase has been shown to be located in the protein bodies of the barley aleurone layers (17).The aleurone layer of rice (Oryza sativa L.) is a storage organ for phosphate in the form of phytin (9, 15) and for lipids (12); it is richer in hemicellulose than the endosperm (4). It is devoid of starch granules in the mature grain (9). Jones (8) Enzyme Extraction. Ten degermed dehulled grains were homogenized at 0 to 4 C at top speed in a VirTis 45 homogenizer for 3 min with 10 ml of 0.1 M tris-HC1 buffer (pH 7.0). The homogenate was centrifuged at 30,000g for 10 min at 4 C, and the crude enzyme was used for the assays, except for assays of lipase and /3-1 ,3-glucanase, which were done on the protein fraction of the crude enzyme precipitating between 0 and 80% saturation with (NH4)2S04. Soluble protein was determined by the Folin phenol reagent (11) corrected for the reading of the tris buffer.Phosphatases and Esterases. The method of Pollard (21) using p-nitrophenyl derivatives was used for the assay of phosphomonoesterase, phosphodiesterase, and esterase. The incubation mixture consisted of the crude extract of degermed seed and substrate equivalent to 0.5 ZImole of p-nitrophenol (mono-phosphate, bisphosphate, and acetate) in 0.1 M acetate buffer (pH 5.0) at 30 C. Incubation time was 15 min for phosphatases and 1 hr for esterase. To stop the reaction, Na,C03 was added, and the amount of p-nitrophenol released was determined colorimetrically at 420 nm. Activities were expressed in ,tmoles of p-nitrophenol formed per hour per seed.Phytase activity was assayed by the method of Mukherji et al. (15) except that incubation time was only 15 min at 30 C at pH 4.0. The amount of phosphate released was measure...

Biochemistry of the Multiple Forms of Glycosidases in Plants

“…Changes in the level of the enzyme have been examined in various plants. The activity increased in the germinating seeds of lupin (85), mung bean (67), Dolichos (57), and rice (223).…”

Aspects of Nitrogen Metabolism in the Rice Seedling

“…Catalase activity was determined by the method of Chance and Maehly (3) as employed by Palmiano and Juliano (23). Incubation time was 1.5 min.…”

Estudio comparativo de la obtención de extractos crudos y semipurificados de lipasa de salvado de arroz

“…La reacción fue interrumpida agregando 4 mL de acetona, seguido de centrifugación a 2000 rpm durante 5 minutos. El sobrenadante fue separado y el p-nitrofenol liberado durante la reacción (de color amarillo) fue cuantificado en un espectrofotómetro a 410 nm con ayuda de una curva de calibrado (Palmiano y Juliano, 1973;Pastore, 1992).…”