protein metabolism in and developing grains of rices ... · plant physiol. (1973) 51, 537-542...

Plant Physiol. (1973) 51, 537-542

Protein Metabolism in Leaves and Developing Grains of RicesDiffering in Grain Protein Content,

Received for publication August 30, 1972

CONSUELO M. PEREZ, GLORIA B. CAGAMPANG, BERNARDITA V. ESMAMA,RUTH U. MONSERRATE, AND BIENvENIDo 0. JuuANOThe International Rice Research Institute, Los Baiios, Laguna, Philippines

ABSTRACT

Four semi-dwarf rices (Oryza sativa L.) differing in percent-age of grain protein, grown in a flooded field receiving basal Nfertilization, had a peak activity of root glutamate dehy-drogenase 4 weeks after transplanting. A lower peak occurredduring panicle formation 10 weeks after transplanting. Thepercentage of N of the active leaf blades was also highest 4 weeksafter transplanting. The activity of nitrate reductase in the leafblades was low and decreased after transplanting.Among the three rices with similar grain yield, the rice with

high percentage of protein tended to translocate more leaf N tothe developing grains than the rices with average grain proteincontent. The leaf blades of the former also had lower rates ofleucine incorporation during grain development but higherprotease activity than leaves of the rices with average proteincontent. Developing grains of the rices with high percentage ofprotein tended to have higher levels of soluble protein, freeamino N, and protease, and a faster rate of leucine incorpora-tion than grains of the 1R8 rice with average percentage ofprotein, regardless of grain yield.

While carbohydrates in the rice grain2 (Oryza sativa L.)result mainly from photosynthesis after flowering, protein Nin the grain is considered by physiologists to be derived mainlyfrom the N already present in the vegetative tissues at flower-ing (8, 16). Among the vegetative tissues of the rice plant theleaf blades have the highest N content (8, 16). Previous studiesat the International Rice Research Institute (IRRI) showedthat the developing grains of lines with high percentages ofprotein had higher amounts of free amino acids than theirlow protein counterparts (2, 4). In lines with high percentageof grain protein the sap entering the panicle generally con-tained larger amounts of free amino acids than the sap in lowprotein lines (2). That indicates a faster breakdown andtranslocation of leaf N to the developing grains of lines withhigh percentage of protein (6, 7).Our study of five rice varieties and lines showed that the

mean protease activity of leaf blades was higher at floweringand during grain development in rices with high yields of grainprotein (6). Field and greenhouse experiments in 1971 showedthat rices with high yields of grain protein had higher per-

' Supported by contract No. PH-43-67-726, National Institute ofArthritis and Metabolic Diseases, National Institutes of Health.

I Rice grain is used to denote dehulled grain or brown rice.

centages and amounts of leaf N from booting to flowering thanrices with low yields of grain protein, and more N translocatedfrom the leaves to the developing grains (7). A high proteinconcentration in leaf blades at booting and flowering wasassociated with a greater decrease in amount of leaf N duringgrain development and with a high level of soluble protein andprotease (7).

Wheats with a high percentage of grain protein have beenreported to translocate leaf N more efficiently to the develop-ing grains than low protein wheats (10). Protease activity inthe leaf is also higher in wheats with high grain protein thanin low protein wheats (23).

Four semi-dwarf rices were grown under flooded field con-ditions and periodically studied for N distribution and bio-chemical factors affecting N metabolism. The rices differ inpercentage and yield of grain protein.

MATERIALS AND METHODS

Seeds of IR8, IR480-5-9, IR160-27-3, and IR1103-15-8(Table I) were steeped in water for 12 hr, germinated for 2days, and grown in a wet bed nursery for 3 weeks. The seedbedwas flooded completely 3 days before transplanting. The seed-lings were transplanted in duplicate 42-m' Agronomy depart-ment plots, two seedlings per hill spaced 20 X 20 cm apart,in a flooded field with 120 kg/ha N applied basally in the1972 dry season. Duplicate two to three hills (not adjacentto missing hills) per plot were sampled weekly, including roots,before 9 AM from 2 weeks after transplanting to maturity. Thesamples were washed, divided into large tillers, small tillers,and roots. The large tillers were separated into active leafblades (second fully expanded leaf from the top [27]), otherleaf blades, leaf sheaths and culms, and panicles. Dry matterand micro-Kjeldahl N (HgO-K,SO, catalyst) (11) were deter-mined for all the plant parts. Brown rice protein was calculatedfrom Kjeldahl N by multiplying by 5.95 (11).

All enzymic assays on vegetative tissues were done on freshsamples except protease, which was done on freeze-drieddeveloping grains and active leaf blades. For the assay ofsoluble glutamate dehydrogenases 1 to 1.5 g of fresh tissuewere frozen and pulverized in the presence of Dry Ice andground in the presence of 1 g of acid-washed sea sand (E.Merck) with 4 ml of cold 0.2 M phosphate buffer, pH 7.8,with 0.01 M GSH in a prechilled mortar for 5 min. The slurrywas squeezed through four layers of cheesecloth and thefiltrate centrifuged at 20,000g for 20 min at 4 C. The super-natant liquid was used for the spectrophotometric GDH assayat 340 nm by the method of Bulen (1). Activity was expressedin ,umoles of NADH oxidized/ming fresh tissue correctedfor the NADH loss in the blank without a-ketoglutarate. Inpreliminary experiments, 4-fold higher GDH activity was

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Plant Physiol. Vol. 51, 1973

IR480:IR8: Nahng

Peta X 'MonDee-gee- S-4/2 Xwoo-gen Taichung

Native 1

110.7 9.14

14.3

99.51114.7

5.14

7.30376

12.6

96.61113.4

4.56

9.45430

IR160:NahngMIonS-4 X

TaichungNative 1

9.57

13.7

105.61214.8

5.26

8.78462

IRI 103:IR8 XChow-sung

10.7

14.3

110.2912.0

3.62

10.8388

LSD(5%)

18.7

2

NS

1.15

2.1

NS

I Transplanting occurred 3 weeks after seeding.2 At 14°c moisture.

observed with NADH as substrate than with NADPH as alsoreported by Saigusa et al. (24).

Sections of leaf blades, leaf sheaths, or roots (about 1 cm

long, 0.5 g) were directly assayed for nitrate reductase by themethod of Jaworski (9): incubation with 10 ml of 0.1 M

phosphate buffer, pH 7.5, with 5% I-propanol and 0.02 M

KNO2 in the presence of two drops of 0.05% chloramphenicolfor 2 hr. A 1-ml portion was mixed with 0.3 ml of % sulf-anilamide in 3 N HCl and 0.02% N-1-naphthylethylenedi-amine HCl for 20 min and absorbance was read at 520 nm.

NR activity was expressed in ,umoles of nitrite formed/hr-gfresh tissue.

Free amino acids were extracted from fresh IR480 roots4 weeks after transplanting by homogenizing in hot 80%ethanol and analysis of the extract was done by a Beckmanamino acid analyzer (2).

Protease activity was determined on ground freeze-driedleaf blades (0.2 g) by extraction for 2 hr at 4 C in 6 ml of 0.01M phosphate buffer, pH 7.5, containing 1 mm dithiothreitol.The mixture was centrifuged at 36,000g for 20 min at 4 C.The supernatant fluid was assayed for protease by the methodof Cruz et al. (4) except that salt-free, freeze-dried hemoglobin(Mann Research Laboratories) was used as substrate insteadof albumin. One milliliter of extract was incubated at 40 Cfor 90 min with 1 ml of 0.1 M phosphate buffer, pH 7.0, and1 ml of 0.5% hemoglobin, and then 1 ml of 10% trichloro-acetic acid was added. After aging for 2 hr at 4 C, the mixturewas centrifuged at 1200g for 15 min at 4 C, and the A2. valueof the supernatant liquid was read. Protease activity was ex-pressed at AA2aw/hr g fresh weight.

Soluble protein was determined in the crude extracts by themethod of Lowry et al. (13) with bovine serum albumin as

standard, and converted to protein N by multiplying by 0.16.Leucine-U-'4C incorporation was done on two sets of 15 leaf

discs, 4.5 mm in diameter, representing 10 leaf blades, usingthe procedure of Cruz et al. (4). The leaf discs were incubatedwith shaking at 37 C for 2 hr with 2 ml of amino acid mixture(0.25 ,tmole each) containing 1 ,uc of leucine-U-'4C (4) after

vacuum infiltration of the discs by evacuating the vials threetimes for a total time of 5 min. The leaf discs were then washedat 0 C three times for 5-min periods, with 10-ml portion of1.0 M phosphate buffer, pH 7.4, and soaked overnight in 3ml of 0.1 M NaOH. The leaf protein was isolated and its radio-activity was determined according to the method of Cruz et al.(4). Activity was expressed in nmoles of leucine/ hr* g freshweight.

Enzymic assays were also done on developing grains at themilky stage. The leucine-U-'4C incorporation assay on develop-ing grains was essentially the method of Cruz et al. (4) exceptthat the grains were dissected lengthwise through the dorsiven-tral axis before incubation. Preliminary studies showed thatdissection increased the incorporation rate 2- to 3-fold. Activitywas expressed in terms of nmoles of leucine/hr g fresh weight.Protease activity was determined on freeze-dried rice powderaccording to the method of Cruz et al. (4). Soluble protein Nwas determined by the method of Lowry et al. (13), total N bymicro-Kjeldahl analysis (11), and free amino N as describedby Cruz et al. (4) but using a modified ninhydrin reagent (15).

RESULTS

Nitrogen Uptake. The rices had similar dry matter produc-tion (Table I). The maximum amounts of N per hill of allfour samples were also similar (Fig. 1). The IR480 line hadthe shortest growth duration and a higher percentage of grainprotein than IR8 (Table I). The IR480 and IR160 lines hadsimilar grain yields as IR8. By contrast, the IR1103 lineshowed a higher percentage of protein but a lower grain yieldthan IR8. It produced fewer panicles per hill than IR160 andhad lighter grains than the other rices. Regardless of grainyield and percentage of grain protein, all four rices yieldedsimilar amounts of grain protein per hectare.

Uptake of N in all samples was most rapid from the 3rdto the 6th week after transplanting. After that N uptakedropped (Fig. 1). Root N was not included in the reported Nvalue because of the difficulty of recovering all the roots inthe older plants. The IR1103 line showed a lower rate of Nuptake than IR8 but it reached about the same maximumamount of N per hill as the other samples by harvest.GDH3 activity per gram of fresh tissue in the 3-week-old

seedlings was higher in the roots than in the leaf blades (TableII). The mean specific activity of GDH in the four sampleswas 1235 nmoles of NADH/minmg protein in roots, 71.2in leaf sheaths, and 4.24 in leaf blades. Similar results werereported by Kanamori et al. (12). Hence, GDH activity wasmeasured mainly in the roots of subsequent samples. Rootsshowed maximum GDH activity 4 weeks after transplantingin the four samples, coinciding with the period of rapid Nuptake (Fig. 1). GDH activity decreased linearly after 4 weeksfrom transplanting but showed a second lower peak 10 weeksafter transplanting.Amino acid analysis of the ethanol extract from IR480 roots

4 weeks after transplanting showed 3-fold molar ratio ofglutamate to aspartate and slightly more than 3-fold ratio ofglutamine to asparagine. The glutamine-glutamate molar ratiowas 1.6 and the asparagine-aspartate ratio was 1.3, indicatingthat a major portion of the glutamate produced by GDH wasconverted to glutamine by glutamine synthetase. Oji andIzawa (18) also reported that barley roots supplied NH4, hadhigher levels of glutamine than glutamate.The NR activity per gram fresh weight in the 3-week-old

I Abbreviations: GDH: glutamate dehydrogenase; NR: nitratereductase.

Table I. Properties at Harvest of Planits of Four Rices Differinzgin Graiin Proteini Conitenzt

Parents

Property

Growth duration1Seeding to panicle

initiation(weeks)

Seeding to flower-ing (weeks)

Plant height (cm)Panicles (No./hill)Total dry matter2

(tons/ha)Brown rice yield2

(tons/ha)Brown rice protein2Brown rice protein

yield (kg/ha)

538 PEREZ ET AL.

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N METABOLISM IN RICE LEAVES AND GRAINS

seedlings at transplanting was higher in leaf blades than in leafsheaths and roots (Table II). Oji and Izawa (17) found a lowerspecific activity of NR in roots than in shoots of 4-week-oldseedlings grown for 3 weeks in nutrient solution containingnitrate N. Leaf NR activity decreased rapidly after transplant-ing (Fig. 1) during the period of nitrogen uptake. Only IR1 103showed a significant increase in NR activity during graindevelopment about 9 weeks after transplanting. Root NRactivity was low and dropped to zero by 10 weeks aftertransplanting (Fig. 1).

Nitrogen Translocation. The N concentration in the activeleaf blades reached a maximum 4 weeks after transplanting forall samples (Fig. 2) after which it dropped progressively. Simi-lar results were obtained for IR8 by Yoshida and Ahn (29).This period of maximum N concentration in active leaf bladescoincides with maximum GDH activity of roots (Fig. 1).Tillering started during this period. That may explain the dropin percentage of N in the large tillers due to translocation to thenew tillers. Maximum tillering occurred 5 to 6 weeks aftertransplanting. The N level in the active leaf blades was similarin the four samples from transplanting except during theperiod shortly before and after the panicle initiation stage inwhich the IR480 line had higher values than IR8 (Fig. 2).The N level of active leaf blades at panicle initiation was 2.6%

Total N (g/hill)

0.4

02 /yv ~~~~VIR1103

Root GDH (pmole NADH min'g' fresh wt)

05g <~

NR (1F mole NO2 hr'I g9 fresh wt0.2 _

V - ~~~~~~~~~~~~Leafbiodeo Root

V. v-,__Jl\ft it j~~~^ n A e D 15 IA I6 a nJ

Weeks after transplanting

FIG. 1. Changes during growth in amount of N per hill, gluta-mate dehydrogenase activity of roots, and nitrate reductase activi-ties of the active leaf blades and roots of four rice samples differingin percentage and yield of protein. LSD (5%) = 0.145 g of N, 0.19Amole of NADH, and 0.05 ,umole of NO2- (leaf).

539

Table 11. Activities of Glutamate Dehlydrogenzase anid NitrateReductase in 21-Day-Old Seedlinigs of Foiur Rice Samples

Enzyme Activity IRS

Glutamate dehydro-genase (pFmolesNADH/min- g

fresh wt)Roots 0.69Leaf sheaths 0.23Leaf blades 0.10

Nitrate reductase(,umole NO-/hr-g fresh wt)

Roots 0.0102Leaf sheaths 0.0063Leaf blades 0.078

IR480

1.050.220.11

0.00900.00500.044

IR160

0.650.120.07

0.00940.00520.067

IR1103 ILSD (5C%)

1.000.280.26

0.00810.00850.074

NSNS

0.12

0.0920NS

0.015

N content of octive ieaf blade (%/Oat 14% H20)6

4-

20 IR8 _ \

0.I I I I1

-4 -2 0 2 4 6 8Weeks from panicle initiation

FIG. 2. Changes in percentage of N of active leaf blades andin dry matter per hill (at 14% moisture) of IR8 and IR480 ricesbefore and after panicle initiation. Panicle initiation occurred 10.7weeks after transplanting in IR8 and 9.14 weeks after transplantingin IR480. LSD (5%) = 0.53% N, and 9.6 g of dry matter per hill.

for IR8, 3.5% for the IR480 line, 3.2% for the IR160 line,and 3.1% for the IR1103 line (LSD (5%) = 0.53%). Themaximum difference between IR8 and the IR480 line inpercentage of N of active leaf blades (1.4 percentage points)occurred shortly after panicle initiation (Fig. 2). The high Ncontent of IR480 and IR160 lines was due mainly to theirlower content of dry matter than IR8 up to 2 weeks afterpanicle initiation (Fig. 2). However, the four rices accumulatedsimilar dry matter at harvest (Table I).


C) 2 4



Piant Physiol. Vol. 51, 1973

Table III. Properties of Fr-esh Leaf Blades anid Roots dutrinzg GrainDevelopment in Folut Rices Differing in Grai,, Proteinl Conztenit

Property of Fresh Tissue IRS 1IR480

A. Active leaf blade (leafNo. 2)

Leucine incorporation 1.9 1.1at flowering (nmoles/hr g)Total N (mg/g) 7.0 10.9

Protease activity at 1 .6 2.7early milky stage'(,A2,,/hr- g)

Total N (mg/g) 7.4 8.1B. Roots

GDH activity at flow- 0.20 0.53ering (,molesNADH/min.g)

Total N (mg/g) 1.25 0.93Soluble protein N 0.083 0.068

(mg/g)

IR160 IR1103 LSD (5%c)

1.4

8.32.3

7.9

0.48

2.1

6.7

1.4

5.2

0.38

1.56 1.520.097 0.108

1 One week after flowering.

Table IV. Properties of Developing Grainis of Fouir Rices Differingin Gr-aini Pr-oteini ConItent

Grain Property per I S I4s R6 R13 LSDGram Fresh XVt IRS jIR4S IRl60 IRl103 (%)

Early milky stage'Protease (AA250/hr) 6.65 8.80 8.80 10.9 2.75Total protein (mg) 36 50 42 58 11Soluble protein (mg) 26.3 30.7 29.3 33.8 NSFree amino N (mg) 0.72 0.90 1.00 1.06 0.26

Milky stage'2Protease (AAq9s/hr) 6.19 8.73 9.38 7.46 3.43Leucine incorporation 2.2 3.8 3.5 4.6 0.7

(nmoles/hr)Total protein (mg) 44 52 50 68 9Soluble protein (mg) 25.0 132.4 32.9 34.0 6.8Free amino N (mg) 0.56 0.73 0.79 0.80 0.17

1 One week after flowering.2 One and one-half weeks after flowering.

Flowering occurred 3.6 weeks after panicle initiation forIR8 and 3.4 weeks for the IR480 line. After flowering theIR480 line showed a greater decrease in percentage of N ofactive leaf blades than IR8. All samples continued to lose leafN even 3 weeks after heading when panicle N was alreadyconstant, presumably due to continued leaf senescence (19).The IR480 line, however, showed the greatest loss of leaf Nshortly before harvest because of its fast leaf senescence. LeafN at harvest was 0.76% for IR8, 0.35% for the IR480 line,0.62% for the IR160 line, and 0.60% for the IR1103 line.However, the flag leaf of the four rices had similar ribulose1 , 5-diphosphate carboxylase activities at flowering and photo-synthetic rates 1 week after flowering (C. M. Perez, unpub-lished data).

In the vegetative stage the leaf blades generally had 3 timesas much N as the leaf sheaths plus culms. During graindevelopment, however, leaf blade N was depleted while theN of leaf sheaths plus culms remained essentially constant.That indicated that the leaf blades were the main source ofleaf N translocated to the developing grains (16). The small

tillers accounted for a mean of 12% of total leaf N in IR8from 4 weeks after transplanting to harvest, 7% in the IR480line, 11% in the IR160 line, and 14% in the IR1103 line.The N content of the small tillers at maturity was 2% of totalleaf N in all rices except in the IRI 103 line where it accountedfor 35% of total leaf N.

Rate of leucine incorporation at flowering tended todecrease with an increase in percentage of N of active leafblade, whereas protease activity tended to increase (Table III).These two properties were highly correlated (r =-0.999**).Leaf properties related to N metabolism were not correlatedwith percentage of grain protein (Table I), since the IRi 103line had similar leaf properties to those of IR8 although thisline had the highest percentage of protein. Among the threerices with similar grain yield, the slower rate of protein syn-thesis and the higher protease level of the leaf blades in IR480as compared to IR8 may account for the greater rate of break-down and translocation of leaf N as amino acids to thedeveloping grains and the higher percentage of protein ofIR480.GDH activity of the roots was higher in the IR480 and

IR160 lines than in IR8 at flowering. It was not related topercentage of total and soluble protein N of root (Table III).The properties of the developing grains at the milky stage

(1 to 1.5 weeks after flowering) were correlated with thedifferences in percentage of grain protein among the foursamples regardless of grain yield (Table IV). Grains with highpercentage of protein, IR480 and IR1 103, had higher proteaseactivities, faster rates of leucine incorporation, and higherconcentrations of soluble protein and free amino N than IR8grains which had average percentage of protein. Similar trendshave been observed earlier by Cruz et al. (4) and Cagampanget al. (2). Results were expressed per gram fresh weight ratherthan per grain because of differences in grain weights amongthe rices. IR8 and the IR480 line had the heaviest grains,followed in order by the IR160 and the IR1 103 lines.

DISCUSSION

Nitrogen Uptake and Translocation. The N in the plantis mainly in protein form and the ratio of protein N to freeamino N is constant in the rice plant during the vegetativestage (19). The four rices in this study differing in percentageof grain protein have similar rates of N uptake. The rices hadsimilar amounts of N per hill at all times (Fig. 1) but only theIR480 line had significantly higher percentage of leaf N thanIR8 immediately after panicle initiation because its dry weight

- was lower than that of IR8 (Fig. 2). In a previous study IR480had both higher amount and percentage of N in leaf bladesthan IR8 (7). Hence, leaf N analysis cannot be used forscreening rice seedlings for percentage or yield of grain protein.The principal difference among the rices studied having

similar grain yield but differing in percentage of grain proteinwas that the rice with high protein yield, IR480, translocatedleaf blade N to the developing grains more efficiently than IR8.More efficient translocation in rices with high percentage ofgrain protein must be due to a high concentration of free aminoacids in the sap which is being translocated to the developinggrains (2) rather than to differences in translocation rates,since similar amounts of sap were collected in the low andhigh protein lines at the midmilky stage (2). Furthermore,wheat plants differing in percentage of grain protein hadsimilar efficiencies of translocation of '4C-labeled amino acidsto the panicle (14).

Johnson et al. (10) found similar levels of leaf N in low andhigh protein wheats. They also concluded that rate of uptakeof N in wheats with high percentage of grain protein is similar

540 PEREZ ET AL.



N METABOLISM IN RICE LEAVES AND GRAINS

to that of low protein wheats but they translocate more leaf Nto the developing grain.

Biochemical Factors Affecting Nitrogen Uptake. GDH activ-ity in the seedlings at transplanting was high in both rootsand leaves and NR activity was noted in both organs (TableII). Since the soil in the seedbed was only flooded 3 daysbefore transplanting, nitrate N may have been present in thesoil and may have explained the low NR and GDH activitiesin the leaf blades which convert nitrate to glutamate via NH4'.The low NR activity even in the seedlings may be explainedby the reported inhibition of NR by NH,' (25). In an earlierexperiment with 4-week-old seedlings from low and high pro-tein rices grown in seedbeds at field capacity, we found thattotal GDH and NR activities of leaf blades and total GDHactivity of roots were positively correlated with the total Ncontent of the three uppermost fully exserted leaf blades (7).

After transplanting, the increase in GDH activity of rootsand the decrease in NR activity of leaf blades are consistentwith NH,' being the main source of N in plant grown inflooded soil. Root GDH activity is induced and increased byNH,+ in rice seedlings (12). The increase in root GDHactivity closely followed N uptake by the plants. The decreasein activity during tillering may have been due in part to thedilution of the root samples from the large tillers by those ofthe small tillers. Although GDH bound to mitochondria hasalso been reported in rice roots, soluble GDH had been shownby Kanamori et al. (12) to predominate and to be more sensi-tive to NH,' supply.The low NR activity in leaves may reflect the trace amount

of nitrate found in rice leaves. The decrease in NR activity inroots with the growth of the rice plant is consistent with theshift of the site of NR activity with plant growth from rootsto leaf blades, considering that nitrate is not as toxic to plantsas NH,+ which had to be detoxified immediately upon uptakein the roots by reductive amination of a-keto acids. Evidentlyroot GDH is a more important enzyme in N uptake by floodedrice than leaf NR. In contrast, in unflooded crops, such aswheat, NR it the key enzyme (3). Leaf NR activity was highestduring the pericd of rapid N uptake at the tillering stage inrice plants grown in culture solution containing nitrate N (17).The growth duration of the IR480 line was shorter than

that of IR8 (Table I) which may explain the higher percentageof grain protein of the IR480 line because soil N level would behigher during grain development. But nutrient solution ex-periments by IRRI physiologists at constant N level (10, 40, or80 ppm up to flowering) showed that the IR480 line alwayshad a higher percentage of grain protein than IR8 at all levelsof nutrient solution N (S. Yoshida and Y. J. Oh, unpublisheddata). Presumably a high percentage of grain protein is not dueto a shorter growth duration alone.

Biochemical Factors Affecting N Translocation. Leaf devel-opment and senescence are known to be under hormonalcontrol (21, 28). The higher percentage of N and faster rateof N depletion in leaf blades of the IR480 rice as comparedto IR8 were associated with a slower rate of protein synthesis(as indexed by the rate of leucine incorporation) and with ahigher protease activity (Table III). Both factors contributeto faster protein degradation in leaf blades of the IR480 rice.Earlier studies, however, indicated that peak protease activityoccurs earlier, at the booting stage, in contrast to rate ofprotein synthesis which peaked at flowering in the flag leafand the active leaf, after which it decreased (7). Hence, theslowing of protein synthesis coincided with the decrease inleaf N content. Singh et al. (26) also reported that older leavesat flowering incorporated less '5N-labeled (NH4)2SO4 into pro-tein than younger leaves. These differences in N translocation

rates had no effect on photosynthetic rates and RuDPcarboxylase activities of the four rices.

Protein accumulation in the developing grain is also underhormonal control (21, 28). During the period of rapid proteinaccumulation (milky stage) the developing grains of rices withhigh percentage of protein had higher rates of protein syn-thesis and higher protease activities than the grains of IR8,the low protein rice (Table IV). Concentrations of solubleprotein and free amino N were also higher in the developinggrains with high protein. These results agree with those ofCruz et al. (4) who used other rices. The high protease activityin the developing grains with high percentage of protein isinteresting since it may reduce the rate of protein accumula-tion. The protease activity was similar to that obtained indegermed seeds of germinating rice (22). However, it ispossible that the protease is located mainly in the embryo andaleurone layers and is not in contact with the protein bodiesof the endosperm, since only 9% of the protease activity inthe mature rice grain is in the endosperm (5).

This study indicates that, at the same fertilizer level, riceswith high percentage of grain protein translocated more leafN in the form of amino acids to the developing grain thanrices with average protein content and with similar grainyield. The developing grains of rices with high percentage ofprotein, regardless of grain protein yield, have higher rates ofprotein accumulation. Of the four rices studied, only the IR480line had a higher percentage of grain protein than IR8 and thesame grain yield as IR8. Addition of N fertilizer during graindevelopment increases the percentage of grain protein by pro-viding an added source of N to the developing grain asindicated by higher levels of NH4, and amino N in the sap(20) and higher amino N levels in the culm and leaf sheathsand the panicles (19).

Differences in growth duration and grain yield among thefour rices studied complicated the interpretation of the results.The study will be repeated as soon as lines from the samecrosses differing mainly in percentage of grain protein becomeavailable in our breeding program.

LITERATURE CITED

1. BULEN-, W. A. 1956. The isolation and characterization of glutamic dehy-drogenase from corn leaves. Arch. Biochem. Biophys. 62: 173-183.

2. CAGA-MPANG, G. B., L. J. CRrz, AND B. 0. JULIANO. 1971. Free amino acidsin the bleeding sap and the developing grain of the rice plant. Cereal Chlem.48: 533-539.

3. CROY, L. I. AND R. H. HAGENIAN. 1970. Relationship of nitrate reductaseactivity to grain protein production in wheat. Crop. Sci. 10: 280-285.

4. CRUZ, L. J., G. B. CAGAMPANG, AND B. 0. JULIANO. 1970. Biochemical factorsaffecting protein accumulation in the rice grain. Plant Physiol. 46: 743-747.

5. HORIGuCHI, T. AN-D K. KITAGISHI. 1969. Changes in protease activ-ity andnitrogen compounds of germinating rice seeds. Studies on rice germ protease(Part 2). Nippon Dojo-Hiryogaku Zasshi 40: 255-259. (English translation(typescript) )

6. International Rice Research Institute. 1971. Annual Report for 1970. LosBafios, Philippines. pp. 13-14.

7. International Rice Research Institute. 1972. Annual Report for 1971. LosBafios, Philippines. pp. 11-14.

8. ISHIZUKA. Y. AND A. TANAKA. 1933. Biochemical studies on the life history ofrice plants. 3. Synthesis and translocation of nitrogen compounds andcarbohydrates. Nippon Dojo-Hiryogaku Zasshi 23: 159-165. (Englishtranslation (typescript) )

9. JAWORSKI, E. G. 1971. Nitrate reductase assay in intact plant tissues. Biochem.Biophys. Res. Commun. 43: 1274-1279.

10. JOHNSON, V. A., J. W. SCHMIDT, AN-D P. J. MATTERN. 1968. Cereal breedingfor better protein impact. Econ. Bot. 22: 16-25.

11. JU-LIANO, B. O., C. C. IGNACIO, V. 'M. PANGAN'IBAN, AND C. NM. PEREZ. 1968.Screening for high protein rice varieties. Cereal Sci. Today 13: 299-301, 313.

12. KAN-AMORI, T., S. KONISHI, AND E. TAKAHASHI. 1972. Inducible formationof glutamate dehydrogenase in rice plant roots by the addition of ammoniato the media. Physiol. Plant. 26: 1-6.

13. LOWRY, 0. H., N. J. RoSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951.

Plant Physiol. Vol. 51, 1973 541



PEREZ ET AL.

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protein metabolism in and developing grains of rices ... · plant physiol. (1973) 51, 537-542...

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