Changes in the content of starch, protein, and RNA and in the activity of their hydrolases in the rice endosperm (Oryza sativa L., variety 1R8) were determined during the first week of germination without added nutrient both in the dark and in the light. Changes were generally more rapid in the dark than in the light. Oxygen uptake and RNase activity started to increase and the root protruded on the second day, followed by the coleoptile on the third day, and the primary leaf on the fourth day. ATP level was at a maximum on the fourth day.The activity of amylases and R enzyme increased progressively, but that of phosphorylase tended to decrease during starch degradation. A new a amylase isozyme band appeared during germination. Glucose was the major product of starch degradation. Sucrose, maltose, maltotriose, raffinose, and fructose were also detected. Protease activity reached a maximum on the fifth or sixth day and closely paralleled the increase in soluble amino N and soluble protein.In embryoless seed halves with 0.12 /tM gibberellin A3, peak protease activity occurred in 2.5 days and peak a amylase activity on the fifth day of incubation. The production of a amylase, protease, and R enzyme was inhibited by 40 uM cyclo. heximide, but only a amylase and R enzyme were inhibited by 20 ,ug/ml actinomycin D.Most studies on biochemical changes in the rice grain (Oryza sativa L.) during germination have been done on samples germinated in the dark (9,25,28). They involved an aspect of either starch or nitrogen metabolism. These studies used different varieties. so they cannot be compared. To obtain a comprehensive picture of the sequence of biochemical events occurring during germination, we studied the changes in activity of degradative enzymes and in their substrates in variety IR8 during the first week of germination both in the light and in the dark. The effect of inhibitors of the synthesis of protein and RNA on levels of a amylase and protease during the germination of embryoless seed halves in the presence of GA3 was also studied.MATERIALS AND METHODS Germination. Rice seeds (variety IR8) were sterilized by soaking them in 1% aqueous NaOCl for 20 min, washed, and 'Supported in part by Contract PH-43-67-726, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health; this paper is in part the M.S. thesis of E. P. P. at the University of the Philippines College of Agriculture. steeped in distilled water for 24 hr. After soaking, the seeds were placed on moist filter paper in covered glass dishes and maintained at 30 C in the dark or in the diffuse light of the laboratory at 25 to 27 C. Germination schedules were staggered to permit harvesting of all the samples at the same time. The hull, roots, shoot, and scutellum were removed, and the degermed grains were freeze-dried, stored at -20 C, and used for all the subsequent analyses.Oxygen Uptake. Oxygen uptake was measured in duplicate from seeds germinated in light for 6 to 86 hr. Ten germinated grains were placed in Warburg fl...

The levels of starch and dextrin, free sugars, soluble protein, and enzymes involved in starch metabolism-a-amylase, ,8-amylase, phosphorylase, Q-enzyme, R-enzyme, and ADPglucose starch synthetases-were assayed in the leaf sheaths and culm of the rice plant (Oryza sativa L., variety IR8) during growth.Starch accumulation in the leaf sheaths reached a maximum 10 to 11 weeks after transplanting, the time of development of the rice panicle. Starch granules have been detected in various tissues of the rice plant during its growth (30,33). The starch of the rice endosperm has been the best characterized of these storage granules (8,14,15). Biochemical studies have been done mainly on the starch of the rice grain (5) and on the starch of the rice leaf chloroplasts (27,29).In contrast to the chloroplast starch, which is transitory, the granules in the parenchyma cells of the leaf sheath and culm accumulate during the vegetative growth of the rice plant and decrease sharply during grain development (30,33,36). Starch in these tissues is reported to be translocated to the panicle, where it contributes to the carbohydrate content of the grain (20,33,36 fully characterized, but this starch tends to have the same amylose-amylopectin ratio in both waxy and nonwaxy rices (12,17).Because of these direct and indirect contributions of the starch of these tissues to grain development and yield, we studied the changes in the properties of starch granules of the leaf sheath and culm and in the levels of enzymes involved in starch metabolism during the growth of the IR8 rice plant. The study was made during the 1970 dry season (JanuaryMay). Yoshida and Ahn (36) The plants were then washed, and only tillers of similar size and age were collected. The late tillers were discarded. The leaf sheaths and culms were removed, immediately cooled to 0 C, and the pooled samples (from nine hills) ground to a powder in the presence of Dry Ice in a Waring Blendor and stored at -20 C. Since the culms were short and small in the early samples before booting, they were not separated from the leaf sheaths. The elongated culms were prepared separately from the leaf sheaths in the samples taken 11, 12, and 14 weeks after transplanting. The panicles were excluded from the culms for all samplings. Leaf blades were discarded since they store very little starch (30,36).Portions of the powdered preparations were homogenized at 0 C (1:3 w/v) with 0.1 M tris-HCl buffer (pH 7.0) for 3 min in a VirTis homogenizer. The homogenate was filtered through four layers of cheesecloth and centrifuged at 27,000g for 10 min at 0 C. The supernatant fluid was decanted and the residue was saved for starch preparation. Protein was precipitated from the supernatant fluid by the addition of solid (NH)-,SO4 to 80% saturation for 30 min at 0 C. Fractions of this suspension were apportioned into several centrifuge tubes and the protein was collected by centrifuging for 10 min at 0 C at 27,000g. Essentially all the enzyme activities assayed were recovered in the precipitat...

The levels of starch, soluble sugars, protein, and enzymes involved in starch metabolism-a-amylase, 3-amylase, pliosphorylase, Q -enzyme, R -enzyme, and starch synthetase -were assayed in dehulled developing rice grains (Oryza sativa L., variety IR8). Phosphorylase, Q-enzyme, and Renzyme had peak activities 10 days after flowering, whereas a-and ,B-amylases had maximal activities 14 days after flowering. Starch synthetase bound to the starch granule increased in activity up to 21 days after flowering. These enzymes (except the starch synthetases) were also detected by polyacrylamide gel electrophoresis. Their activity in grains at the midmilky stage (8-10 days after flowering) was determined in five pairs of lines with low and high amylose content from different crosses. The samples had similar levels of amylases, phosphorylase, R-enzyme, and Q-enzyme. The samples consistently differed in their levels of starch synthetase bound to the starch granule, which was proportional to amylose content. Granule-bound starch synthetase may be responsible for the integrity of amylose in the developing starch granule.Most starch biochemists believe that the starch synthetases (ADP-and UDP-glucose-starch 4-glucosyltransferases) are the enzymes involved in starch synthesis (2,20). Some investigators believe that phosphorylase is also a synthetic enzyme and that the starch synthetase functions to protect the amylose molecule formed with phosphorylase and starch synthetase from being transformed to amylopectin through the action of Q-enzyme (1, 13). Still others propose a multiple pathway synthesis of starch (4).Another unsolved problem in starch biosynthesis is the genetic integrity of amylose in the granule of nonwaxy starches. Previous work on developing rice and corn grains showed that starch synthetase bound to the starch granule occurs mainly in nonwaxy granules (3,20 Q-enzyme (a-1 ,4-glucan:a-1 ,4-glucan 6-glycosyltransferase or branching enzyme), and R-enzyme (amylopectin 6-glucanohydrolase or debranching enzyme)-during grain development in the rice variety IR8. Lines from the same cross differing in amylose content were used to compare enzyme activities in relation to amylose synthesis. The use of such lines grown in the same crop reduces complicating environmental and genetic factors which accompany studies in which different varieties are compared. MATERIALS AND METHODSSamples of developing rice grains (Oryza sativa L., variety IR8) were obtained from the experimental field of the Institute at 7, 10, 14, 21, and 28 days after flowering and immediately stored at 0 C. They were dehulled by hand prior to analysis. The grain weight was determined for each sample.Samples of lines differing in amylose content were grown in a Mylar house in pots containing 6 kg of air-dried soil. One day before transplanting, 10 g of (NH4)2S04 and 8 g Na3PO4 were added to the soil, the soil was mixed well, and the pots were flooded. Four 10-day-old seedlings were transplanted per pot and kept under continuous flooding. Panicles were t...

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...

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