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...

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