Plant Physiol. (1970) 46, 743-747

Biochemical Factors Affecting Protein Accumulation inthe Rice Grain'

Received for publication March 23, 1970

LOURDES J. CRUZ,2 GLORIA B. CAGAMPANG, AND BIENVENIDO 0. JULIANOInternational Rice Research Institute, Los Banios, Laguna, Philippines

ABSTRACT

Rice grains (Oryza sativa L.) from three varieties andthree pairs of lines with different protein content werecollected at 4-day intervals from 4 to 32 days after flowering.The samples were analyzed for protein, free amino nitrogen,ribonucleic acid, protease activity, and ribonuclease ac-tivity. In addition, the capacity of the intact grain to incor-porate amino acids was determined for the three pairs oflines. The maximal level of free amino nitrogen and thecapacity of the developing grain to incorporate amino acidswere consistently found to be higher in the samples withthe high protein content in the mature grain. The ribo-nucleic acid content of the grain tended to be higher in thehigh protein samples.

The protein content of brown rice (Oryza sativa L.) rangesfrom about 5 to 17% (15, 16, 18). The level of protein in themature grain has been found to be consistently higher in somevarieties than in others (18). However, the protein content of avariety may vary by as much as 7% (5). Application of nitrogenfertilizer to the rice plant at the ear initiation or flowering stagesand low levels of solar radiation during grain development maycontribute to a higher percentage of protein in the rice grain.Genetic and environmental factors were found to have similareffects on the changes of amino acid composition and ratio ofprotein fractions in rice grains (12, 18).As part of a program to breed higher protein rice, we studied

the various biochemical factors which may contribute to thedifferences in the rate and extent of protein accumulation in thedeveloping rice grain. Varieties and lines differing in mature grainprotein were used for the study. Knowledge of the causes ofdifferences in grain protein accumulation among varieties mayalso be useful in studying how environmental factors affect theprotein content of the grain.

MATERIALS AND METHODS

Pot Experiments. For this study, a low protein, an intermediateprotein, and a high protein rice variety, were grown during the

1 Supported in part by Contract PH-43-67-726 from the NationalInstitute of Arthritis and Metabolic Diseases, National Institutes ofHealth. We acknowledge the assistance of B. P. Gapud on the chemicaldeterminations, and of the Varietal Improvement Department of In-ternational Rice Research Institute for providing the samples.

2Present address: Biochemistry Department, University of the Philip-pines, College of Medicine, Herran Street, Manila, Philippines.

1968 wet season. One plant was grown per pot containing 7.5 kgof soil, 10 g of (NH4)2SO4, 4 g of KCl, and 4 g of Ca(HPO4)2.To simulate field conditions, the pots were buried to the brim insoil in a screened concrete bed with a spacing of 40 X 40 cmbetween plants. Three planting dates for each variety were used.Eighteen plants were planted at each date. The planting dateswere scheduled so that the three varieties flowered on the samedate, thus subjecting the plants to the same weather conditionsduring grain development. The concrete bed was continuouslyflooded. All plants were treated regularly with insecticides.

Field Experiments. Pairs of dwarf lines from the F4 plant gen-eration of IR8 crosses with four high protein varieties (RikutoNorin 20, Chow Sung, Omirt 39, Crythroceros Korn) were se-lected to cover a wide range of protein content. The plants weregrown in the 1968-69 dry season at the Institute experimentalfarm. They received 80 kg/hectare N at planting time. Each linewas represented by four plants selected from a row of 20 plantsspaced 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 examineddaily and tagged when they flowered because the flowering datesof the spikelets of a single panicle, as well as of the panicles ofdifferent tillers of a single plant ranged over several days. Thegrains were cooled to 0 C in an ice bath immediately after samplingand were stored at -20 C until used for analysis. However,freshly collected samples were used for assaying synthetic capac-ity because this activity was found to decrease progressively at-20 C.Moisture Determination. One hundred grains kept frozen over

Dry Ice were dehulled by hand, then placed in tared 15-ml testtubes. The weight loss during freeze-drying for 48 hr was takenas the moisture content.

Homogenization. The grains from the moisture determinationwere soaked overnight at 0 C in 13.5 ml of 0.05 M tris buffer (pH7.5) with 0.5 M KCl (the volume of 100 freeze-dried grains hadpreviously been found to be about 1.5 ml). The soaked grainswere homogenized at 0 C for 3 min with a VirTis 45 homogenizerat top speed. Six milliliters of the homogenate were set aside tobe used for the determination of protein content, nucleic acid,and free amino nitrogen. About 9 ml were centrifuged at 500gfor 15 min at 4 C, and the supernatant fluid was reserved forassaying ribonuclease activitiy.

Chemical Determinations. Six milliliters of the crude homog-enate (equivalent to 40 grains) were treated with 6 ml of cold,20% (w/v) trichloroacetic acid for 1 hr at 0 C and then frac-tionated according to the scheme shown in Figure 1.

Free Amino Nitrogen. The amount of soluble amino nitrogenin extract I was determined by using the ninhydrin method de-scribed by Moore and Stein (26) as modified by Moore (25). Totest tubes containing 16.2 mg of Na2CO3, 0.50 ml of extract Iwas added. The solutions were treated with 1.0 ml of ninhydrinreagent (25). The test tubes were covered with glass marbles and

743

www.plantphysiol.orgon May 2, 2020 - Published by Downloaded from Copyright © 1970 American Society of Plant Biologists. All rights reserved.

CRUZ, CAGAMPANG, AND JULIANO

Suspend in 6.0 ml cold20% TCA for 1 holr

Centrifuge (1200g,10 min, 40)

Supernatant fluid

6ml (ExtractI)A for (soluble amino N)

L200g.

?Pt

Suspend in 10 ml 5% TCAHead at 900 for 30 minCentrifuge (1200g, 10 min, 250)

+FPpt

Reextract with 10r5% TCA at 900

Ppt(protein)

.nl

n1 ~ICombined supernatant

fluid (Extract II)(nucleic acid)

FIG. 1. Scheme for the separation of the nitrogenous fractions ofthe rice grain. TCA: Trichloroacetic acid.

were heated in boiling water for 15 min and then cooled for5min in cold water. The colored solutions were diluted 10-foldwith 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 pre-

cipitate was removed by filtering through Whatman No. 541paper. A portion of the filtrate (0.1 ml) was diluted with 0.9 ml ofwater, and the protein concentration was determined by themethod of Lowry et al. (20) with crystalline bovine plasma al-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 throughWhatman No. 42 paper. A portion of the filtrate was diluted 10-fold with distilled water, and the absorbance (A) was determinedat 260 and 280 nm. Yeast RNA (Nutritional Biochemicals Corp.,reagent grade) and bovine plasma albumin in 0.5% trichloroace-tic acid were used as the standards. The blank used for this assay

contained an equivalent amount of trichloroacetic acid. The A260of the nucleic acids of the trichloroacetic acid extract was calcu-lated by correcting for the A26o/A28o ratio of the contaminatingproteins (22, 32), and the corresponding nucleic acid concentra-tions were determined from a standard RNA curve. The concen-

tration of DNA in extract II was determined by the method ofBurton (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 DNAassay. To estimate the amount of DNA which would give thesame color reaction as dAMP, the amount of dAMP was multi-plied by twice the average molecular weight of the deoxynucleo-tide residues. The factor 2 was used because only one-half of thesugar residues (those combined with purines) in the DNA reactwith the diphenylamine reagent (3).

Ribonuclease Activity. The assay used to determine RNaseactivity was the method of Tuve and Anfinsen (31) as modifiedby Wilson (33) and Cagampang (4). One unit of RNase activitywas defined as the amount of enzyme which under the conditionsof the assay produced a 0.1 increase in light absorbance (A260)of the solution (4).

Protease Activity. One hundred dehulled grains were soaked in13.5 ml of 0.01M sodium phosphate buffer (pH 7.5) containing0.005M cysteine. The grains were homogenized for 3 min andcentrifuged at500g for 15 min at 0 C, and the supernatant fluidwas assayed for protease activity according to the method ofBeevers (2) with modification. One milliliter of the enzyme prep-aration was incubated with 1.0 ml of1 % bovine albumin (Cal-biochem, B grade) dissolved in water and 1.0 ml of 0.2M sodiumphosphate buffer (pH 6.5) at 40 C for 90 min. For each sample,duplicate tubes plus a zero time control were prepared. The reac-tion was stopped with 1.0 ml of 20%o trichloroacetic acid, and theprecipitate was aged for at least1 hr at 0 C. The mixture wascentrifuged at 1200g for 15 min at 4 C. A portion of the super-natant fluid was diluted 4-fold with distilled water, and the ab-sorbance at 280 nm was determined with a 1-cm cuvette. One unitof protease activity was defined as the amount of enzyme thatproduced a 0.1 increase inlight absorbance of the undiluted tri-chloroacetic acid supernatant fluid obtained under the conditionsof the assay. No activity was observed in the absence of cysteine.The protease activity of grain extracts with 0.2M sodium phos-phate buffer was found to be 1.3 times the activity of the 0.01Mbuffer extracts.

Leucine-U-'4C Incorporation by Intact Grains. Fifty fresh grainswere dehulled and incubated in a tightly closed vial with 5 ml of0.1M potassium phosphate buffer containing 0.5,umoleof L-leu-cine-U-'4C (Calbiochem, 5,uc, at least 94% pure by paper chro-matography) and 0.25,imole each of the following L-aminO acids.alanine, arginine, asparagine, aspartic acid, cysteine, glutamicacid, glutamine, glycine, histidine, lysine, methionine, phenyl-alanine, proline, serine, threonine, tryptophan, tyrosine, valine,and isoleucine. After 2 hr in a water bath shaker at 37 C, the vialwas immediately placed in crushed ice. Then the incubationmedium was decanted and the grains were washed three timeswith 20 ml of 0.1M potassium phosphate buffer (pH 7.4). Eachtime, the wash solution was shaken for at least 5 min in an icebath. The washed grains were homogenized for 3 min with 24.2ml of potassium phosphate buffer. The homogenate was centri-fuged at 500g for 15 min. A 0.1-ml portion of the supernatantfluid was plated on filter paper disks (Whatman No. 5271-S10,3 mm) mounted on stainless steel pins, and dried in an oven at45 C. Another0.1-ml portion was applied on the disk. The diskwas dried and then treated as described by Mans and Novelli(21). For blanks, 50 grains were swirled with the cold incubationmedium, decanted, washed, homogenized, and analyzed in thesame way as the samples. As standards, filter paper disks towhich 0.2 ml of blank had been applied and which had beentreated according to the procedure of Mans and Novelli wereplated with known amounts of incubation media or of standardL-leucine-U-'4C.

RESULTSThree Varieties Differing in Protein Content. The first series of

experiments was done primarily to study the changes duringripening in a variety with low protein (Peta), intermediate pro-tein (IR8), and high protein (A2 torzs). The spikelets filled mostrapidly from 4 to 16 days after flowering. Filling continued up to20 days. From then until maturity, the dry weight of the dehulledgrain was almost constant. The dehulled grain weight of maturePeta was 21 mg; of IR8, 20 mg; and of A2 torzs, 24 mg. We ob-served that from 20 days after flowering up to maturity, A2 torzsweighed about 20% more than IR8 and 14% more than Peta.The moisture content decreased from about 80% at 4 days toless than 20% at 32 days. Similar results for dry matter and watercontent 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

6.0 ml crude homogenate(40 grains)

PptRe suspend incold 5% TC.15 min

Centrifuge110 min, 4

Supernatantfluid

(discard)

744 Plant Physiol. Vol. 46, 1970

www.plantphysiol.orgon May 2, 2020 - Published by Downloaded from Copyright © 1970 American Society of Plant Biologists. All rights reserved.

PROTEIN ACCUMULATION IN RICE GRAIN

CP

z

0

C

.E0

.0

0I

10

5

0

0 4 8 12 14

.0

01

0

:01

cm

01

n

0

-

z

40

20

4 8 12 16 20 32Da 32 o

Days after f lowering

FIG. 2. Changes in protein content and in factors related to protein metabolism in the developing rice grain of three varieties differing inprotein content (IR8, 10.4% protein; Peta, 10.5% protein; A2 torzs, 12.1% protein).

days after flowering (Fig. 2). The protein content per grainleveled off after the 16th day. Although the absolute amount ofprotein increased rapidly between 4 and 16 days after flowering,the actual percentage decreased perhaps because starch accumu-lated faster at this period of grain development. The same trendfor protein nitrogen was observed by Jennings and Morton (13)in developing wheat endosperm and by Palmiano et al. (29) inrice. The protein contents (dry basis) of brown (dehulled) ricefor 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. Hoshi-kawa (9) found that cell division was completed 9 days afterflowering for a Japanese variety. Since tropical rice ripens fasterthan Japanese rice, our data expressed on a per grain basis shouldbe proportional to a per cell basis for 8-day or older grain. Mat-sushita (22) reported that the RNA content of the rice grainreached a maximum about 14 days after flowering.The pattern of changes in the levels of protein and RNA re-

ported in this study have previously been reported in grains ofmaturing rice (29, 30), wheat (14), and corn (10). Studies ondeveloping wheat grain (7) have shown that during the period ofrapid increase in endospermal protein, a related increase in RNAcontent occurs. Oota (27) reported that the rate of protein ac-cumulation in growing cotyledons of maturing bean seeds is alinear function of the concentration of the total RNA in thetissues. Matsushita (23) reported that the ratio of RNA to pro-tein decreased from 0.07 to 0.02 during the course of ripening ofwheat grains.

In our experiments the amount of soluble amino nitrogen in-creased initially and reached a maximum between the 8th and the12th days after flowering. It subsequently dropped to about 25%of the maximal value. It remained at this low level from the 20thday up to maturity. The relationship between the curves for

soluble amino nitrogen and protein content was consistent sincefree amino acids are the precursors of protein in the grain. Themaximal accumulation of proteins in the grain and the maximallevel of free amino nitrogen occur at the same time. The highprotein sample (A2 torzs) exhibited higher levels of solubleamino nitrogen than low protein samples, an indication that inthe grain the level of free amino nitrogen is a function of the totalN. Palmiano et al. (29) reported that nonprotein nitrogen was themajor fraction of the total N in the 4-day-old grain of IR8 butnot in the mature grain. A similar trend was observed in develop-ing rice (1) and wheat (13).Maximal protease activity occurred between the 8th and 12th

days of grain development. In contrast, RNase activity reached apeak between the 12th and the 16th days (Fig. 2). Our resultsshow that peak RNase and protease activities occur after rapidRNA and protein syntheses. The relationship between RNA andRNase 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 wasrelated to high RNase activity. No relationship between theRNase activity and the protein content of the grain was obtained.The high protein grain tended to have a higher protease activitythan the low protein grain. However, the activities ofprotease andRNase in the developing rice grain were low. We had to activateprotease with cysteine to obtain even the low values reported(Fig. 2). Hence, the protease values we obtained are mainly thoseof the inactive enzyme. Kaminski and Bushuk (19) similarlyfound extremely low activities of proteases in wheat flour bystarch-gel electrophoresis. Ozaki and Horiguchi (28) still foundlow activities in mature rice grain even after treatment withcysteine. Germinated rice, however, exhibited high proteaseactivities (8, 28).A comparison of the three rice varieties revealed that the RNase

activities were practically the same. The maximal levels of free

._

01o0

CP_1.0

._

0

0

100

01

01

z:r

50

'I

0

64

a-

.-

-P-lant Physiol. Vol. 46, 1970 745

A2 tOrzs

to

i4 8 12 16 20

2n

P%

www.plantphysiol.orgon May 2, 2020 - Published by Downloaded from Copyright © 1970 American Society of Plant Biologists. All rights reserved.

CRUZ, CAGAMPANG, AND JULIANO

_ 20

E

_ 1.0

_c

0c 920

*2

-

u60

c.6o

4.z

40

20

10

za

6

3¢ 2

0

.c 2.0

2

0

O.-

it. 0.4

40

Q

3I

30

20

10

0 4 8 12 16 20 32 0 4 8 12 16 20 32

Days after flowering Days after flowering

FIG. 3. Changes in protein content and in factors related to protein metabolism in the developing F5 rice grain of two lines from IR8 X Ri-kuto Norin 20 differing in protein content (IRI100-12-12, 11.7% protein; IR1 100-128-1, 8.6% protein).

amino nitrogen, RNA, and protease activity were greater for thehigh protein variety, A2 torzs, than for either Peta or IR8. Thevalidity of such a comparison may be questioned owing to differ-ences between A2 torzs and Peta or IR8. Compared with the twovarieties, A2 torzs has a lower yield, a shorter growth duration, avery different plant type, and a heavier grain. However, the higherlevel 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 theF4 generation of IR8 crosses with high protein varieties werechosen to cover a wide range of protein content. The lines were

subjected to the analyses mentioned above for the developinggrains. In addition, the lines were analyzed for the L-leucine-U-'4Cincorporation. The protein content of the dehulled mature grainsranged from 8.49 to 11.7%, dry basis. The stage of ripening dur-ing which the factors were at a peak varied among the lines. Asexpected, the higher the protein content, the greater was thecapacity of the grain to incorporate L-leucine-U-14C (maximalvalues ranged from 67.3 to 136 m,ug/2 hr), and the higher was themaximal level of free amino nitrogen (a range of 6.0-14.3 u.g).The high protein samples tended to have higher maximal levelsof RNA than the low protein samples. Maximal activity ofRNase ranged from 24 to 36 units per grain. Maximal proteaseactivity ranged from 1.2 to 1.5 units per grain. These values weresimilar to those observed for the three varieties.The members of one pair of lines, IR1100-12-12 and IR1100-

128-1, were similar in growth duration and yield. The changestaking place during the ripening of these two lines are shown inFigure 3. The dry weights of single grains were 19.5 mg forIR1100-12-12 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 maximallevels of soluble amino nitrogen, the rates of amino acid incor-poration, and the protease activities were higher for the highprotein line than for the low protein line. As was observed for thethree varieties, the maximal levels of soluble amino nitrogenoccurred at the same time as the maximal increase in proteincontent. Furthermore, the rate of amino acid incorporation wasalso highest at this stage. The high protein line incorporated 1.7times as much '4C as the low protein counterpart. This indicatesthat, although protein synthesis in the two varieties begins at thesame time, the rates of synthesis are different. It is this differencethroughout grain development that caused different amounts ofprotein to accumulate in the grains of the high protein and lowprotein lines. The protease activity assayed at pH 6.5 was higherfor the high protein line than for the low protein line. Only minordifferences in the maximal RNA content and RNase activitieswere observed between the two lines.

DISCUSSION

Our data show that the two factors affecting protein accumula-tion during the first 2 weeks of grain development-the level offree amino acids and the capacity of the intact grain to incor-porate amino acids-are correlated with the sample differencesin protein content of the mature rice grain. Presumably, the higbprotein grains have higher levels of all the nitrogenous fractionsincluding RNA.The effect of the level of free amino acids on protein accumula-

tion is quite obvious. Amino acids are the precursors of storageproteins in the rice grain. Hence, a higher level of these aminoacids will contribute to a faster and greater accumulation ofprotein in the so-called protein bodies (30), even if the levels ofenzymes involved in protein synthesis are the same in all grains.

"7-- - .

o---o IR1100-12-12 /-.*IRIIOO-128-1//-o/_°

.r I I { 1 1a

N'/

///

le --b,,

.-4 0

I"

746 Plant Physiol. Vol. 46, 1970

www.plantphysiol.orgon May 2, 2020 - Published by Downloaded from Copyright © 1970 American Society of Plant Biologists. All rights reserved.

PROTEIN ACCUMULATION IN RICE GRAIN

The greater capacity for amino acid incorporation in the de-veloping grain of samples with higher protein content providesinteresting biochemical explanations. These results imply a higherconcentration of enzymes involved in the protein synthesis. Thelevels of these enzymes are probably under genetic control. Sinceprotein synthesis involves a series of enzymatic reactions, onecannot readily envision a relationship between the concentrationof free amino acids and the enzymes involved in protein synthesissuch as exists between nitrate concentration and nitrate reductase.Incorporation rates with intact seeds are very slow, and moreefficient techniques are needed to determine whether this is reallyan 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 thegrain. This may be due specifically to a higher level of messengerRNA which carries the informationfor the particular protein to besynthesized, although the other types of RNA-ribosomal andtransfer-are also known to be essential for protein synthesis.Thus, one may expect to find different types of messenger RNAto predominate at different stages of grain development wherevarying fractions of grain proteins are being synthesized. How-ever, Natori (11) found no significant difference in the base com-position of the total RNA in the panicle during the booting andmilky 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 areexpected. During the maturation process, the equilibrium be-tween 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 thedeveloping grain, rather than the capacity of the grain to incor-porate amino acid. In addition, high protein content in the grainbrought about by nitrogen fertilization is found to increase onlythe storage proteins-glutelin and prolamin (5). This means thenthat the enzyme protein per grain is constant throughout thedevelopment period.

In summary, only the maximal level of the free amino nitrogenand the capacity for amino acid incorporation of the developinggrain consistently correlated with the protein content of maturerice grains. In the plant itself, the free amino acid pool of thegrain is continuously being replenished by amino acids from thesap. Among the lines tested, differences in the level of aminonitrogen in the grain may reflect differences in the efficiency ofsoil nitrogen absorption by the root system or in the rate oftranslocation ofamino nitrogen. Our preliminary study shows thatin the high protein lines the sap that is translocated into thepanicles 10 days after flowering has a higher content of solubleamino acids than the sap in low protein counterparts. The as-paragine and glutamine fractions of the total nitrogen in the sapof high protein lines were also higher.

LITERATURE CITED

1. AIMI, R. AND MURAKAMI, T. 1959. Paper-chromatographic survey of protein andamino 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 ofgerminating pea seeds (Pisum sativum). Phytochemistry 7: 1837-1844.

3. BURTON, K. 1956. A study of the conditions and mechanisms of the diphenylaminereaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J.62: 315-323.

4. CAGAMPANG, G. B. 1968. Ribonuclease activity of maize (Zea mays L.) endospermduring 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. CerealChem. 43: 145-155.

6. DALBY, A. AND I. DAviEs. 1967. Ribonuclease activity in the developing seed ofnormal 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. Na-ture 196: 967-969.

8. HORIGUCHI, T. AND K. KITAGISHI. 1969. Changes in protease activity and nitrogencompounds of germinating rice seeds. Nippon Dojo-Hiryogaku Zasshi 40: 255-259.

9. HOSHIKAWA, K. 1967. Studies on the development of endosperm in rice. 1. Processof endosperm tissue formation. Nippon Sakumotsu Gakkai Kiji 36: 151-161.

10. INGLE, J., D. BEITZ, AND R. H. HAGEMAN. 1965. Changes in composition duringdevelopment 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 contentof rice. 2nd Annu. Rep., National Institutes of Health Contract No. PH-43-67-726, pp. 2-3.

13. JENNINGs, A. C. AND R. K. MORTON. 1963. Changes in carbohydrate, protein, andnon-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 indeveloping 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 proteincontent, amylose content, and alkali digestibility of rice varieties in Asia. Philip-pine Agr. 48: 234-241.

17. JULiANo, B. O., G. M. BAUTISTA, J. C. LUGAY, AND A. C. REYES. 1964. Studies onthe 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. Screen-ing for high protein varieties. Cereal Sci. Today 13: 299-301, 331.

19. KAMINisKI, E. AND W. BUSHUK. 1969. Wheat proteases. I. Separation and detectionby starch gel electrophoresis. Cereal Chem. 46: 317-324.

20. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Proteinmeasurement 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 theradioactive amino acid into protein by a filter paper disk method. Arch. Bio-chem. Biophys. 94: 48-53.

22. MATSUSHITA, S. 1958. Studies on the nucleic acids in plants. I. Nucleic acid con-tents 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 thenucleic 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 amountsof RNA and ribonuclease activity in the grains during the ripening process ofwheat. Mem. Res. Inst. Food Sci. Kyoto Univ. 19: 1-5.

25. MOORE, S. 1968. Amino acid analysis: Aqueous dimethylsultoxide as solvent forthe ninhydrin reaction. J. Biol. Chem. 243: 6281-6283.

26. MOORE, S. AND W. H. STEIN. 1954. A modified ninhydrin reagent for the photo-metric 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. NipponDojo-Hiryogaku Zasshi 36: 255-259.

29. PALMIANO, E. P., A. M. ALMAZAN, AND B. 0. JULIANO. 1968. Physicochemicalproperties 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. Composi-tion and endosperm structure of developing and mature rice kernel. CerealChem. 45: 225-235.

31. TuvE, T. W. AND C. B. ANFINSEN. 1960. Preparation and propertiesofspinach ribo-nuclease. 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. Bio-chim. Biophys. Acta 68: 177-184.

Plant Physiol. Vol. 46, 1970 747

www.plantphysiol.orgon May 2, 2020 - Published by Downloaded from Copyright © 1970 American Society of Plant Biologists. All rights reserved.