Biochemical Factors Affecting Protein Accumulation in the Rice Grain

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bumin (Calbiochem, A grade) as the standard. RNA and DNA. ExtractII was diluted to 25 ml with 5% tri- chloroacetic acid, then filtered at room temperature ...

Plant Physiol. (1970) 46, 743-747

Biochemical Factors Affecting Protein Accumulation in the Rice Grain' Received for publication March 23, 1970

LOURDES J. CRUZ,2 GLORIA B. CAGAMPANG, AND BIENVENIDO 0. JULIANO International Rice Research Institute, Los Banios, Laguna, Philippines 1968 wet season. One plant was grown per pot containing 7.5 kg of soil, 10 g of (NH4)2SO4, 4 g of KCl, and 4 g of Ca(HPO4)2. Rice grains (Oryza sativa L.) from three varieties and To simulate field conditions, the pots were buried to the brim in three pairs of lines with different protein content were soil in a screened concrete bed with a spacing of 40 X 40 cm collected at 4-day intervals from 4 to 32 days after flowering. between plants. Three planting dates for each variety were used. The samples were analyzed for protein, free amino nitrogen, Eighteen plants were planted at each date. The planting dates ribonucleic acid, protease activity, and ribonuclease ac- were scheduled so that the three varieties flowered on the same tivity. In addition, the capacity of the intact grain to incor- date, thus subjecting the plants to the same weather conditions porate amino acids was determined for the three pairs of during grain development. The concrete bed was continuously lines. The maximal level of free amino nitrogen and the flooded. All plants were treated regularly with insecticides. capacity of the developing grain to incorporate amino acids Field Experiments. Pairs of dwarf lines from the F4 plant genwere consistently found to be higher in the samples with eration of IR8 crosses with four high protein varieties (Rikuto the high protein content in the mature grain. The ribo- Norin 20, Chow Sung, Omirt 39, Crythroceros Korn) were senucleic acid content of the grain tended to be higher in the lected to cover a wide range of protein content. The plants were high protein samples. grown in the 1968-69 dry season at the Institute experimental farm. They received 80 kg/hectare N at planting time. Each line was represented by four plants selected from a row of 20 plants spaced 20 cm apart. The distance between rows was 30 cm. Sample Collection. Grain samples were collected from 4 to 32 days after flowering at 4-day intervals. Spikelets were examined daily and tagged when they flowered because the flowering dates The protein content of brown rice (Oryza sativa L.) ranges of the spikelets of a single panicle, as well as of the panicles of from about 5 to 17% (15, 16, 18). The level of protein in the different tillers of a single plant ranged over several days. The mature grain has been found to be consistently higher in some grains were cooled to 0 C in an ice bath immediately after sampling varieties than in others (18). However, the protein content of a and were stored at -20 C until used for analysis. However, variety may vary by as much as 7% (5). Application of nitrogen freshly collected samples were used for assaying synthetic capacfertilizer to the rice plant at the ear initiation or flowering stages ity because this activity was found to decrease progressively at and low levels of solar radiation during grain development may -20 C. contribute to a higher percentage of protein in the rice grain. Moisture Determination. One hundred grains kept frozen over Genetic and environmental factors were found to have similar Dry Ice were dehulled by hand, then placed in tared 15-ml test effects on the changes of amino acid composition and ratio of tubes. The weight loss during freeze-drying for 48 hr was taken protein fractions in rice grains (12, 18). as the moisture content. As part of a program to breed higher protein rice, we studied Homogenization. The grains from the moisture determination the various biochemical factors which may contribute to the were soaked overnight at 0 C in 13.5 ml of 0.05 M tris buffer (pH differences in the rate and extent of protein accumulation in the 7.5) with 0.5 M KCl (the volume of 100 freeze-dried grains had developing rice grain. Varieties and lines differing in mature grain previously been found to be about 1.5 ml). The soaked grains protein were used for the study. Knowledge of the causes of were homogenized at 0 C for 3 min with a VirTis 45 homogenizer differences in grain protein accumulation among varieties may at top speed. Six milliliters of the homogenate were set aside to also be useful in studying how environmental factors affect the be used for the determination of protein content, nucleic acid, protein content of the grain. and free amino nitrogen. About 9 ml were centrifuged at 500g for 15 min at 4 C, and the supernatant fluid was reserved for MATERIALS AND METHODS assaying ribonuclease activitiy. Chemical Determinations. Six milliliters of the crude homogPot Experiments. For this study, a low protein, an intermediate enate (equivalent to 40 grains) were treated with 6 ml of cold, protein, and a high protein rice variety, were grown during the 20% (w/v) trichloroacetic acid for 1 hr at 0 C and then fractionated according to the scheme shown in Figure 1. 1 Supported in part by Contract PH-43-67-726 from the National Free Amino Nitrogen. The amount of soluble amino nitrogen Institute of Arthritis and Metabolic Diseases, National Institutes of in extract I was determined by using the ninhydrin method deHealth. We acknowledge the assistance of B. P. Gapud on the chemical scribed by Moore and Stein (26) as modified by Moore (25). To determinations, and of the Varietal Improvement Department of In- test tubes containing 16.2 mg of Na2CO3, 0.50 ml of extract I ternational Rice Research Institute for providing the samples. 2Present address: Biochemistry Department, University of the Philip- was added. The solutions were treated with 1.0 ml of ninhydrin reagent (25). The test tubes were covered with glass marbles and pines, College of Medicine, Herran Street, Manila, Philippines. ABSTRACT

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0 ml crude homogenate (40 grains)

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(soluble amino N)

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Protease Activity. One hundred dehulled grains were soaked in 13.5 ml of 0.01M sodium phosphate buffer (pH 7.5) containing 0.005 cysteine. The grains were homogenized for 3 min and centrifuged at 500g for 15 min at 0 C, and the supernatant fluid was assayed for protease activity according to the method of Beevers (2) with One milliliter of the enzyme preparation was incubated with 1.0 ml of1 % bovine albumin (Calbiochem, B grade) dissolved in water and 1.0 ml of 0.2M sodium phosphate buffer (pH 6.5) at 40 C for 90 min. For each sample, duplicate tubes plus a zero time control were prepared. The reaction was stopped with 1.0 ml of trichloroacetic acid, and the precipitate was aged for at least1 hr at 0 C. The mixture was for 15 min at 4 C. A portion of the supercentrifuged at natant fluid was diluted 4-fold with distilled water, and the absorbance at 280 nm was determined with a 1-cm cuvette. One unit of protease activity was defined as the amount of enzyme that absorbance of the undiluted triproduced a 0.1 increase in chloroacetic acid supernatant fluid obtained under the conditions of the assay. No activity was observed in the absence of cysteine. The protease activity of grain extracts with 0.2M sodium phosphate buffer was found to be 1.3 times the activity of the 0.01M buffer extracts. Leucine-U-'4C Incorporation by Intact Grains. Fifty fresh grains were and incubated in a tightly closed vial with 5 ml of 0.1 potassium phosphate buffer containing 0.5 ,umoleof L-leucine-U-'4C (Calbiochem, 5 at least 94% pure by paper chroeach of the following L-aminO acids. matography) and 0.25 alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and isoleucine. After 2 hr in a water bath shaker at 37 C, the vial was immediately placed in crushed ice. Then the incubation medium was decanted and the grains were washed three times with 20 ml of 0.1 M potassium phosphate buffer (pH 7.4). Each time, the wash solution was shaken for at least 5 min in an ice bath. The washed grains were homogenized for 3 min with 24.2 ml of potassium phosphate buffer. The homogenate was centriA 0.1-ml portion of the supernatant for 15 fuged at fluid was plated on filter paper disks (Whatman No. 5271-S10, 3 mm) mounted on stainless steel pins, and dried in an oven at 45 C. Another on the disk. The disk portion was was dried and then treated as described by Mans and Novelli (21). For blanks, 50 grains were swirled with the cold incubation in the medium, decanted, washed, homogenized, and analyzeddisks to same way as the samples. As standards, filter paper which 0.2 ml of blank had been and which had been treated according to the procedure of Mans and Novelli were plated with known amounts of incubation media or of standard L-leucine-U-'4C.

M

Supernatant fluid

Ppt

Plant Physiol. Vol. 46, 1970

Suspend in 10 ml 5% TCA Head at 900 for 30 min Centrifuge (1200g, 10 min,

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light

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(nucleic acid) FIG. 1. Scheme for the separation of the nitrogenous fractions of

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heated in boiling water for 15 min and then cooled for5 min in cold water. The colored solutions were diluted 10-fold with 50% ethanol and their absorbances were read at 570nm. The standard used was L-leucine (Eastman Organic Chemicals). Protein. The residue from the trichloroacetic acid washings was suspended in enough1 N NaOH to make a total volume of 25 ml. The mixture was aged for 30 min, heated at 100 C for 10 min, mixed, and then allowed to stand overnight. The gelatinous precipitate was removed by filtering through Whatman No. 541 paper. A portion of the filtrate (0.1 ml) was diluted with 0.9 ml of were

water, and the protein concentration was determined by the method of Lowry et al. (20) with crystalline bovine plasma albumin (Calbiochem, A grade) as the standard. RNA and DNA. Extract II was diluted to 25 ml with 5% trichloroacetic acid, then filtered at room temperature through Whatman No. 42 paper. A portion of the filtrate was diluted 10fold with distilled water, and the absorbance (A) was determined at 260 and 280 nm. Yeast RNA (Nutritional Biochemicals Corp., reagent grade) and bovine plasma albumin in 0.5 % trichloroacetic acid were used as the standards. The blank used for this assay contained an equivalent amount of trichloroacetic acid. The A260 of the nucleic acids of the trichloroacetic acid extract was calculated by correcting for the A26o/A28o ratio of the contaminating proteins (22, 32), and the corresponding nucleic acid concentrations were determined from a standard RNA curve. The concentration of DNA in extract II was determined by the method of Burton (3), and the amount of RNA was estimated by difference. Disodium deoxyadenosine 5'-monophosphate hydrate (dAMP, Calbiochem, A grade) was used as the standard for the DNA assay. To estimate the amount of DNA which would give the same color reaction as dAMP, the amount of dAMP was multiplied by twice the average molecular weight of the deoxynucleotide residues. The factor 2 was used because only one-half of the sugar residues (those combined with purines) in the DNA react with the diphenylamine reagent (3). Ribonuclease Activity. The assay used to determine RNase activity was the method of Tuve and Anfinsen (31) as modified by Wilson (33) and Cagampang (4). One unit of RNase activity was defined as the amount of enzyme which under the conditions of the assay produced a 0.1 increase in light absorbance (A260)

of the solution (4).

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dehulled

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500g

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RESULTS Three Varieties Differing in Protein Content. The first series of experiments was done primarily to study the changes during ripening in a variety with low protein (Peta), intermediate protein (IR8), and high protein (A2 torzs). The spikelets filled most up to rapidly from 4 to 16 days after flowering. Filling continued 20 days. From then until maturity, the dry weight of the dehulled grain was almost constant. The dehulled grain weight of mature 20 mg; and of A2 torzs, 24 mg. We obPeta was 21 mg; of served that from 20 days after flowering up to maturity, A2 torzs weighed about 20% more than IR8 and 14% more than Peta. The moisture content decreased from about 80% at 4 days to less than 20 % at 32 days. Similar results for dry matter and water content have been reported for IR8 by del Rosario et al. (30), and for a Japanese variety by Matsushita (23). The increase in protein content was most rapid from 4 to 16

IR8,

-P-lant Physiol. Vol. 46, 1970

PROTEIN ACCUMULATION IN RICE GRAIN

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days after flowering (Fig. 2). The protein content per grain leveled off after the 16th day. Although the absolute amount of protein increased rapidly between 4 and 16 days after flowering, the actual percentage decreased perhaps because starch accumulated faster at this period of grain development. The same trend for protein nitrogen was observed by Jennings and Morton (13) in developing wheat endosperm and by Palmiano et al. (29) in rice. The protein contents (dry basis) of brown (dehulled) rice for this particular crop were 10.4% for IR8, 10.5% for Peta, and 12.1% for A2 torzs. RNA synthesis (Fig. 2) occurred mainly during the first 8 days after flowering and preceded maximal protein synthesis. Hoshikawa (9) found that cell division was completed 9 days after flowering for a Japanese variety. Since tropical rice ripens faster than Japanese rice, our data expressed on a per grain basis should be proportional to a per cell basis for 8-day or older grain. Matsushita (22) reported that the RNA content of the rice grain reached a maximum about 14 days after flowering. The pattern of changes in the levels of protein and RNA reported in this study have previously been reported in grains of maturing rice (29, 30), wheat (14), and corn (10). Studies on developing wheat grain (7) have shown that during the period of rapid increase in endospermal protein, a related increase in RNA content occurs. Oota (27) reported that the rate of protein accumulation in growing cotyledons of maturing bean seeds is a linear function of the concentration of the total RNA in the tissues. Matsushita (23) reported that the ratio of RNA to protein decreased from 0.07 to 0.02 during the course of ripening of wheat grains. In our experiments the amount of soluble amino nitrogen increased initially and reached a maximum between the 8th and the 12th days after flowering. It subsequently dropped to about 25% of the maximal value. It remained at this low level from the 20th day up to maturity. The relationship between the curves for

soluble amino nitrogen and protein content was consistent since free amino acids are the precursors of protein in the grain. The maximal accumulation of proteins in the grain and the maximal level of free amino nitrogen occur at the same time. The high protein sample (A2 torzs) exhibited higher levels of soluble amino nitrogen than low protein samples, an indication that in the grain the level of free amino nitrogen is a function of the total N. Palmiano et al. (29) reported that nonprotein nitrogen was the major fraction of the total N in the 4-day-old grain of IR8 but not in the mature grain. A similar trend was observed in developing rice (1) and wheat (13). Maximal protease activity occurred between the 8th and 12th days of grain development. In contrast, RNase activity reached a peak between the 12th and the 16th days (Fig. 2). Our results show that peak RNase and protease activities occur after rapid RNA and protein syntheses. The relationship between RNA and RNase could explain the slight decrease in the RNA content. This is consistent with what was found in developing corn (4, 6, 10): a rapid decrease in the amount of endospermal RNA was related to high RNase activity. No relationship between the RNase activity and the protein content of the grain was obtained. The high protein grain tended to have a higher protease activity than the low protein grain. However, the activities of protease and RNase in the developing rice grain were low. We had to activate protease with cysteine to obtain even the low values reported (Fig. 2). Hence, the protease values we obtained are mainly those of the inactive enzyme. Kaminski and Bushuk (19) similarly found extremely low activities of proteases in wheat flour by starch-gel electrophoresis. Ozaki and Horiguchi (28) still found low activities in mature rice grain even after treatment with cysteine. Germinated rice, however, exhibited high protease activities (8, 28). A comparison of the three rice varieties revealed that the RNase activities were practically the same. The maximal levels of free

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Plant Physiol. Vol. 46, 1970

CRUZ, CAGAMPANG, AND JULIANO "7-_ 20 E _

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amino nitrogen, RNA, and protease activity were greater for the high protein variety, A2 torzs, than for either Peta or IR8. The validity of such a comparison may be questioned owing to differences between A2 torzs and Peta or IR8. Compared with the two varieties, A2 torzs has a lower yield, a shorter growth duration, a very different plant type, and a heavier grain. However, the higher level of protein and soluble amino nitrogen of A2 torzs was more than can be accounted for by its heavier grain weight. Three Pairs of Lines Differing in Protein Content. To find plants which were closer genetically, three pairs of lines from the F4 generation of IR8 crosses with high protein varieties were chosen to cover a wide range of protein content. The lines were subjected to the analyses mentioned above for the developing grains. In addition, the lines were analyzed for the L-leucine-U-'4C incorporation. The protein content of the dehulled mature grains ranged from 8.49 to 11.7%, dry basis. The stage of ripening during which the factors were at a peak varied among the lines. As expected, the higher the protein content, the greater was the capacity of the grain to incorporate L-leucine-U-14C (maximal values ranged from 67.3 to 136 m,ug/2 hr), and the higher was the maximal level of free amino nitrogen (a range of 6.0-14.3 u.g). The high protein samples tended to have higher maximal levels of RNA than the low protein samples. Maximal activity of RNase ranged from 24 to 36 units per grain. Maximal protease activity ranged from 1.2 to 1.5 units per grain. These values were similar to those observed for the three varieties. The members of one pair of lines, IR1100-12-12 and IR1100128-1, were similar in growth duration and yield. The changes taking place during the ripening of these two lines are shown in Figure 3. The dry weights of single grains were 19.5 mg for IR1100-12-12 and 18.0 mg for IR1100-128-1. The protein content of the dried 32-day-old grains was 11.7% for the high protein line (IR1100-12-12) and 8.56%7, for the low

protein line (IR1100-128-1). Of the factors studied, the maximal levels of soluble amino nitrogen, the rates of amino acid incorporation, and the protease activities were higher for the high protein line than for the low protein line. As was observed for the three varieties, the maximal levels of soluble amino nitrogen occurred at the same time as the maximal increase in protein content. Furthermore, the rate of amino acid incorporation was also highest at this stage. The high protein line incorporated 1.7 times as much '4C as the low protein counterpart. This indicates that, although protein synthesis in the two varieties begins at the same time, the rates of synthesis are different. It is this difference throughout grain development that caused different amounts of protein to accumulate in the grains of the high protein and low protein lines. The protease activity assayed at pH 6.5 was higher for the high protein line than for the low protein line. Only minor differences in the maximal RNA content and RNase activities were observed between the two lines. DISCUSSION Our data show that the two factors affecting protein accumulation during the first 2 weeks of grain development-the level of free amino acids and the capacity of the intact grain to incorporate amino acids-are correlated with the sample differences in protein content of the mature rice grain. Presumably, the higb protein grains have higher levels of all the nitrogenous fractions including RNA. The effect of the level of free amino acids on protein accumulation is quite obvious. Amino acids are the precursors of storage proteins in the rice grain. Hence, a higher level of these amino acids will contribute to a faster and greater accumulation of protein in the so-called protein bodies (30), even if the levels of enzymes involved in protein synthesis are the same in all grains.

Plant Physiol. Vol.

46, 1970

PROTEIN ACCUMULATION IN RICE GRAIN

The greater capacity for amino acid incorporation in the developing grain of samples with higher protein content provides interesting biochemical explanations. These results imply a higher concentration of enzymes involved in the protein synthesis. The levels of these enzymes are probably under genetic control. Since protein synthesis involves a series of enzymatic reactions, one cannot readily envision a relationship between the concentration of free amino acids and the enzymes involved in protein synthesis such as exists between nitrate concentration and nitrate reductase. Incorporation rates with intact seeds are very slow, and more efficient techniques are needed to determine whether this is really an important factor in vivo. The slightly higher level of RNA in the high protein samples may also contribute to the faster accumulation of protein in the grain. This may be due specifically to a higher level of messenger RNA which carries the informationfor the particular protein to be synthesized, although the other types of RNA-ribosomal and transfer-are also known to be essential for protein synthesis. Thus, one may expect to find different types of messenger RNA to predominate at different stages of grain development where varying fractions of grain proteins are being synthesized. However, Natori (11) found no significant difference in the base composition of the total RNA in the panicle during the booting and milky stages of the rice plant. The low activities of the RNase and protease and their lack of relation to the accumulation of protein in the ripening grain are expected. During the maturation process, the equilibrium between synthesis and degradation is shifted toward the former. How does nitrogen nutrition affect protein accumulation in the rice grain? Probably, it changes the level of free amino acid in the developing grain, rather than the capacity of the grain to incorporate amino acid. In addition, high protein content in the grain brought about by nitrogen fertilization is found to increase only the storage proteins-glutelin and prolamin (5). This means then that the enzyme protein per grain is constant throughout the development period. In summary, only the maximal level of the free amino nitrogen and the capacity for amino acid incorporation of the developing grain consistently correlated with the protein content of mature rice grains. In the plant itself, the free amino acid pool of the grain is continuously being replenished by amino acids from the sap. Among the lines tested, differences in the level of amino nitrogen in the grain may reflect differences in the efficiency of soil nitrogen absorption by the root system or in the rate of translocation of amino nitrogen. Our preliminary study shows that in the high protein lines the sap that is translocated into the panicles 10 days after flowering has a higher content of soluble amino acids than the sap in low protein counterparts. The asparagine and glutamine fractions of the total nitrogen in the sap of high protein lines were also higher. LITERATURE CITED 1. AIMI, R. AND MURAKAMI, T. 1959. Paper-chromatographic survey of protein and amino acids in rice plant during ripening. Nippon Sakumotsu Gakkai Kiji 27: 412-416. 2. BEEVERS, L. 1968. Protein degradation and proteolytic activity in the cotyledons of germinating pea seeds (Pisum sativum). Phytochemistry 7: 1837-1844. 3. BURTON, K. 1956. A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 62: 315-323.

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4. CAGAMPANG, G. B. 1968. Ribonuclease activity of maize (Zea mays L.) endosperm during development. M.S. thesis. Purdue University, Lafayette, Ind. 5. CAGAMPANG, G. B., L. J. CRUZ, S. G. EsPIRITU, R. G. SANTIAGO, AND B. 0. JuLANo. 1966. Studies on the extraction and composition of rice proteins. Cereal Chem. 43: 145-155. 6. DALBY, A. AND I. DAviEs. 1967. Ribonuclease activity in the developing seed of normal and opaque-2 maize. Science 155: 1573-1575. 7. GRAHAM, J. S. D., A. C. JENNINGS, R. K. MORTON, B. A. PALK, AND J. K. RAISON. 1962. Protein bodies and protein synthesis in developing wheat endosperm. Nature 196: 967-969. 8. HORIGUCHI, T. AND K. KITAGISHI. 1969. Changes in protease activity and nitrogen compounds of germinating rice seeds. Nippon Dojo-Hiryogaku Zasshi 40: 255259. 9. HOSHIKAWA, K. 1967. Studies on the development of endosperm in rice. 1. Process of endosperm tissue formation. Nippon Sakumotsu Gakkai Kiji 36: 151-161. 10. INGLE, J., D. BEITZ, AND R. H. HAGEMAN. 1965. Changes in composition during development and maturation of maize seeds. Plant Physiol. 40: 832-835. 11. International Rice Research Institute. 1967. 1966 Annual Report. The Institute, Los Bafios, Laguna, Philippines. pp. 34-35. 12. International Rice Research Institute. 1969. Improvement of the protein content of rice. 2nd Annu. Rep., National Institutes of Health Contract No. PH-43-67726, pp. 2-3. 13. JENNINGs, A. C. AND R. K. MORTON. 1963. Changes in carbohydrate, protein, and non-protein nitrogenous compounds of developing wheat grain. Aust. J. Biol. Sci. 16: 318-331. 14. JENNINGs, A. C. AND R. K. MORTON. 1963. Amino acids and protein synthesis in developing wheat endosperm. Aust. J. Biol. Sci. 16: 384-394. 15. JULIANO, B. 0. 1966. Physicochemical data on the rice grain. Int. Rice Res. Inst. Tech. Bull. 6. 16. JULIANO, B. O., E. L. ALBANO, AND G. B. CAGAMPANG. 1964. Variability in protein content, amylose content, and alkali digestibility of rice varieties in Asia. Philippine Agr. 48: 234-241. 17. JULiANo, B. O., G. M. BAUTISTA, J. C. LUGAY, AND A. C. REYES. 1964. Studies on the physicochemical properties of rice. J. Agr. Food Chem. 12: 131-138. 18. JuLIANO, B. O., C. C. IGNACIO, V. M. PANGANIBAN, AND C. M. PEREZ. 1968. Screening for high protein varieties. Cereal Sci. Today 13: 299-301, 331. 19. KAMINisKI, E. AND W. BUSHUK. 1969. Wheat proteases. I. Separation and detection by starch gel electrophoresis. Cereal Chem. 46: 317-324. 20. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. 21. MANS, R. J. AND G. D. NovEw. 1961. Measurement of the incorporation of the radioactive amino acid into protein by a filter paper disk method. Arch. Biochem. Biophys. 94: 48-53. 22. MATSUSHITA, S. 1958. Studies on the nucleic acids in plants. I. Nucleic acid contents of cereal and pulse seeds and their nucleic acid containing fraction. Mem. Res. Inst. Food Sci. Kyoto Univ. 14: 14-23. 23. MATSUSHITA, S. 1958. Studies on the nucleic acids in plants. II. Variations of the nucleic acid contents of wheat and rice grains during ripening processes. Mem. Res. Inst. Food Sci. Kyoto Univ. 14: 24-29. 24. MATSUSHITA, S. 1959. On the protein formation and the changes of the amounts of RNA and ribonuclease activity in the grains during the ripening process of wheat. Mem. Res. Inst. Food Sci. Kyoto Univ. 19: 1-5. 25. MOORE, S. 1968. Amino acid analysis: Aqueous dimethylsultoxide as solvent for the ninhydrin reaction. J. Biol. Chem. 243: 6281-6283. 26. MOORE, S. AND W. H. STEIN. 1954. A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J. Biol. Chem. 211: 907-913. 27. OOTA, Y. 1964. RNA in developing plant cells. Annu. Rev. Plant Physiol. 15: 17-36. 28. OZAKI, K. AND T. HORIGUCHI. 1965. Studies on rice germ protease. 1. Nippon Dojo-Hiryogaku Zasshi 36: 255-259. 29. PALMIANO, E. P., A. M. ALMAZAN, AND B. 0. JULIANO. 1968. Physicochemical properties of developing and mature rice grain. Cereal Chem. 45: 1-12. 30. ROSARIO, A. R. DEL, V. P. BRIONES, A. J. VtDAL, AND B. 0. JUUANO. 1968. Composition and endosperm structure of developing and mature rice kernel. Cereal Chem. 45: 225-235. 31. TuvE, T. W. AND C. B. ANFINSEN. 1960. Preparation and propertiesofspinach ribonuclease. J. Biol. Chem. 235: 3437-3441. 32. WARBURG, 0. AND W. CHRISTIAN. 1942. Isolation and crystallization of enolase. Biochem. Z. 310: 384-421. 33. WILSON, C. M. 1963. Chromatographic separation of ribonucleases in corn. Biochim. Biophys. Acta 68: 177-184.

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