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Pliysiol. (1967) 42, 327-332

Biosynthesis of Starch in ' T. Nomura, N. Nakayama, T. Murata2, and T. Akazawa Faculty of Agriculture, Nagoya University, Nagoya, Japan Received November 16, 1966.

Summa71l(lry. The enzymic synthesis of ADP- and UDP-gluicose by chloro- pyrophosphorylase of and has been demonstrated by paper chromatographic techniques. In both tissues, the activity of UDP-glucose-pyrophos- phorylase was much higher than ADP-glucose-pyrophosphorylase. Glycerate-3-phos- phate, phosphoenolpyruvate and fruictose-1,6-diphosphate did not stimuilate ADP-glucose formation by a pyrophosphorylation reaction. The major for UDP-glucose utilization appears to be the synthesis of either or sucrose-P. On the other hand, a specific precursor role of ADP-gluicose for synthesizing chloro- plast starch by the ADP-glucose-starch transglu-cosylase reaction is suipported by the coupled system of ADP-gltucose-pyrophosphorylase and transglucosylase, iso- lated from chloroplasts. None of the glycolytic intermediates stimtulated the glulcose transfer in the enz me sequenice of reaction system employed.

Suicrose is generally considered to be a major of interest from the view of the photosynthetic carbon assimilation produict in chloroplasts of photo- carbon . This paper deals with the en- synthesizing plant organ. This produict is even- zymic synthesis of ADP-glucose and UDP-glicose. tually transported in the form of non-reduicing Starch synthetase associated with the to other plant parts (1, 2, 7). On starch, which operates chiefly throtugh ADP-glucose the other hand, in the chloroplasts of some di- pathway, was also studied. cotyledons and photosynthetic green , prom- inent starch formation has been demonstrated (1, 2). Materials and Methods Starch of this specific character is conventionally called assimilation starch, which shouild more appro- priately be termed as chloroplast starch. In con- Grozwing of . Both bean and rice were trast to reserve starch, chloroplast starch exists in grown in in a greenhouise for about 14 to 15 transitory form and is readily uitilized as an energy days. The bean was at fifth and sixth stage souirce in the metabolism of leaf tissuies. This is and rice at third to fouirth leaf stage. observed in the diurnal pattern of its synthesis Iso(lationt of Chloroplasts Preparation. Freeze- and breakdown (32). drie(l chloroplasts were prepared uising the non- aquleouis methodl Early on of Stocking (30). studies the syinthesis of chloroplast Preparation of tile starch received mulch attention, in particuilar, the Solutble Entzymiie. A) Beani role of chloroplastic (1, 19, 29). leaves (60 g) were homogenized in a WVaring However, the cuirrent view on starch biosynthesis, Blendor with 210 ml of 50 mair btuffer through the ntucleotide has (pH 7.0) containing 0.5 % iso-ascorbate. After pathway, led squeezing the homogenate throuigh cloth uis to reexamine the enzymic mechanism of the (8 lavers), process. Muirata and Akazawa (21) and Ohosh the filtrate was centrifuiged at 15,000 and X g for 20 minutites. The solu-tion was adjtusted Preiss (11, 12) have reported the specific role to pH 5.0 with acetic of ADP-gltucose starch transglutcosylase in starch 1 N and centriftuged again biosynthesis iunder conditions where the UDP- at 15,000 X g for 10 minuLtes. The resuilting pre- glucose pathway was totally cipitate was dissolved in 60 ml of 10 mm K-phos- inactive. Since sul- phate buffer (pH 7.0), made to 30 % saturation crose and sucrose-P synthesis in both leaf tissuies by adlding and chloroplasts involving UDP-glulcose has been crystallinie (NH4)2SO, and centrifuged reported by several workers (3, 4, 6, 8, 14, 20), the at 10,000 X g for 10 minuttes to remove most of interplay of the 2 different the soluble (fraction-I-protein). The sulper- nlucleotidle stugars is natant fraction was made to 55 % satulration by adding crystalline (NS\H,)2SO4, and centrifulged at 10,000 X g for 10 minuites. The precipitate was 'Supported in part by a Research Gra'rt of United dissolved in 1.0 ml of distilled , dialyzed States Public Hez.?th Service No. AI 10792-1. 2 Present address: Tohoku National Agrictiltural Ex- against w'ater for abouit 2 houirs in a cold room periment Station, Morioka, Japan. (20), and then uised as an enzyme souirce. 327 328 P) Rice leaves (60 g) were first frozen with MgSO4, 9.0; UDP-glucose-'4C, 2.2 (1.9 X 10-2 lry ice, then grotun(d wvith the same btuffer as that l'c, 4.7 X 104 cpm); -6-P, 100; enzyme usedI for extracting soluible enzyme of bean leaves. preparation, 200 ,ul in a total volume of 1.26 ml. Stubsequent steps were essentially the same as that The mixture was incu,bated for 60 minutes at 370. employed for bean leaves. At the end of this incuibation period, the reaction Prepar(ationi of Clhloroplast Starch. Bean leaves mixtture was treated by the method uised previouslN (50 g) were harveste(d at midday and immediately and applied to a of Dowex-1 anion exchange homogenized to obtain the starch granules as resin (23). Carrier -P (2-4 mg) were added reported previously (21). Greenish white starch to the eltuate, and aliquiots uised for the measuirement samples were used for the enzyme assay. of radioactivity of suicrose (26) and total sutgar Enzynte Assay. A) ADP-glucose and UDP- (9). gltucose pyrophosphorylase. Reaction mixture con- Reagents. All the chemical reagenits used were tained (in Amoles): glycine buffer (pH 8.5), 5.0; commercial prodtucts, and ADP-glucose-'4C, UDP- MgSO4, 0.1; EDTA, 0.25; NaF, 2.5; GSH, 0.5; glucose-'4C, and glucose-14C-1-P were purchased ATP, 0.84 or UTP, 0.54; glucose-14C-1-P, 0.5 (2.7 from International Nuclear Corporation, California. 10-2 C, 5.9 X 104 cpm), either chloroplasts preparation, 1.0 mg (eqv. 0.01 mg chlorophvll and Results and Discussion 0.28 mg protein) or solutble enzyme, 50 1A (ca 10.2 mg protein/ml), and H.O, 50 ul in a total voltume .SyWthesis of ADP-glutcose and UDP-Pglucose. of 90 ,ul. The reaction was continued for 60 min- Enzvnmic synithesis of ADP-gluicose ad(I UDI)'- uttes at 37°. After adding 2.0 ml of 80 % (V/V) ethalnol, the whole reaction mixtuire was centrifuged, ancl the residuie was washed twice with . G-1-P + ATP The whole extract was evaporated in vacuio at 20, 3000 anld dissolved in 100 AI of water. One half of the finial eltuate was applie(d to anI acid-washe(d What- mani No. 1 paper ancl chromatographed as reported 2000 - previotusly (22, 23). The radioactivity was deter- minied in a Packard liquiid scintillation cotunter. B-) ADP-glutcose and UDP-glucose starch trans- gltcos\lase. The reactioll mixture containiedl (in 0.~?1000- gumoles): glycine buffer (pH 8.5), 5.0; MgSO4, u, 1.0; EDTA, 0.25; ADP-glticose IC, 0.16 (1.2 X 1()-0ju2 , 2.7 X 104 cpm), or UDP-glucose-14C, 0.16 5; (8.2 X 10- yc, 1.8 X 104 cpm); chloroplast starch 10 20 30 40 g-ranuiles, 5.0 mg; and H20, 50 gl in a total voluime u 300OfG-1-P+UTP of 90 ,ul. The method of assaying 14C-incorpora- tionl inito starch was the same0 as that reported ThA previously (22, 23). C) Couipling system of pyrophosphorylase an(l transglucosvlase. The composition of the reaction mixtuire Nvas essentially the same as that for the pvrophosphorylase sy-stem, except that either soluble enzyme preparatioin (50 ALl) or chloroplasts (1.0 mgn) was added, in a(lditioin to the chloroplast starch sample (5.0 mg). I)) Sticrose-synthetase. The soluble enzvme as explainedl above (A) was tise(l for the sucrose synthesis experiment in the following reaction mixtuire (in umoles): tris huffer (pH 7.5), 30; ADPG,UDPG G-1-P G MgSO, 0.9; NaF, 20; fruictose or frtuctose-6-P, FIG. 1. 1.(0: UDP-glucose or ADP-gluicose, 0.25; ADP-gluicose anid UDP-glucose sxnltlhesis by andl en- r-ice chloroplasts. Reaction mixture conttained (in zyme preparationi, 20 A1 in a total voltume of 130 ul. ,umoles) glycine buffer (pH 8.5), At the 5.0; MIgSO, 1.0; en(d of incuigbationl, the stucrose formation was FDTA, 0.25; GSH, 0.5; ATP, 0.84 or UTP, 0.54; gli- assayed by the resorciinol method of Roe (26). cose-i4C-1-P, 0.5 (2.7 X 10-2 ,uc, 5.9 X 104 cpm) Io1t Exchange Columniiii Chro matography. Dow- chlioroplast preparation 1.0 mg; and H^O, 50 ul in a total ex-1 anion exchange coluimn chromatography was volumes of 90 14l. Reaction was continued for 60 minuttes carried ouit to analyze the formation of sucrose-P at 370, and the 80 % (V/V) etbanol eluate of the re- prodtuced in actant w-as analyzed by paper chromatography. Abbre- the following reaction mixture (in viations tris butffer (pH /7.4), 400; used are: ADPG, ADP-glucose; UDPG, UDP- .Imoles) NaF, 400; glutcose; G-1-P, glucose-1-P; anid G, glucose. NOMURA ET AL.-CHLOROPLAST STARCH 329 glucose by pyrophosphorylase residing in chloro- plast preparations of rice leaves was demonstrated by paper chromatography (fig 1). Essentially identical paper chromatogram was obtained by the bean chloroplast preparation, btut the enzyme ac- tivity of the bean plant was much higher. There M- exist discrepancies in the literatture concerning the localization of UDP-glucose-pyrophosphorylase in chloroplasts. Opposed to the initial notion on the 1001X( localization of UDP-glucose-pyrophosphorylase out- side chloroplasts (28), recent experiments of Bird et al. (3) have shown the presence of enzyme in chloroplasts isolated from tobacco leaves by non- 0 aqtueouis method. The specific activity (20 mumoles 50 UDP-gluicose formed/mg protein/60 minutes) of the °u S soluble enzyme preparation used in our present experiment showed nearly the same valtue (24 mlmoles UDP-glucose formed/mg protein/60 min- utes') of the whole chloroplasts preparation obtained by the noni-aqtueouis method, suipporting a confined TIME IN MINUTES localization of the enzyme in chloroplasts. This finding is indeedI in accordance with the restults of FIG. 3. Sucrose and sucrose-P syniithesis by soluble Bird et al. (3). \Vhole chloroplasts and soluible enzxyme of rice leaves. Reaction mixture contained (in ,umoles): tris buffer (pH 7.5), 30; MgSO4, 0.9; NaF, enzyme preparationi of both rice and bean system 20; fructose or fructose-6-P, 1.0; UDP-glucose or ADP- showed a markedl activity of the glycolytic trans- glucose, 0.25; and soluble enzyme preparation, 20 ,Al in a total volume of 130 ,u. Fructose + UDP-glucose. o) 0; fructose-6-P + UD,P-glucose, 0 0; and fructose + ADP-glucose, A AN. Sucrose was as- sayed by the method of Roe (23).

formation of glulcose-l-P to gltucose-6-P and( frulc- tose-6-P. This transformation was muich less in the soluible enzyme system, however. The presence of phosphoglutcomuitase and phosphohexoisomerase in chloroplasts has been reported by Bird et al. (3). It has been claimed by Preiss and his associates (11, 24, 27) that some glycoly-tic intermediates, in- cluding glycerate-3-P, P-enolpyruivate, fructose-l- 6-P, stimulate chloroplastic and bacterial ADP- glulcose-pvrophosphorylase. WN e teste(d these com- pouind(s withouit anyt positive effect, regardlless of the var;ed coincenitrationi raniges of the campolinl( s (0.5-5.0) mat) and -variouis inculbation time intervals (15-180 min). Syn1tthesis of Sucrosc and Sutcrose-P. In an1 attempt to examine the role of UDP-gluicose, as seen in the preceeding experiment, the suicrose- synthesizing system was tested. There is milch debate concerning the site of suicrose syinthesis in chloroplasts (3, 14, 16, 31). Several inivestigations favor the idea that there is enzymic synthesis of 20 30 suicrose and suicrose-P in chloroplasts. Our analysis 0 clearly indicates the presence of UDP-glucose-sui- crose (suicrose-P) transgluicosylationi, and also ADP UDP G-l-P G ADP-glulcose-suicrose transgluicosvlatioin, reactions in rice leaf tissuies FIG. 2. ADPG and UDPG synthesis by soluble pyro- (fig 3). The prodtuction of phosphorylase of rice leaves. Experimental condition was sucrose-P was rigorously established by ion-ex- the samle as that of figure 1, except 50 ,l of soluble change coluimn chromatography (fig 4), althouigh enzyme preparation of rice leaves xX-as used. Same abbre- it cannot be ascertained from this stuidv alone viation,; as that for fizure 1 were employed. whether fruictose or frulctose-6-P reacts predomi- 330 PLANT PHYSIOLOGY nantly in initact leaf tissues. It is probable that earlier than stucrose-P is not known, although this suicrose-P is enzymically hydrolyzed to free suicrose fractioni has often been detectedI in 14C-feeding by a specific suicrose-P phosphatase (15). The experiments (23). natuire of the minor radioactive compound eltutable Starch Sv'ttliesis. Resuilts of the preferenltial utilization of ADP-glucose by starch synlthetase, associated with the chloroplast starch granuiles, conforms with ouir preliminarv experiment (21) (table I). Miany later workers obtained similar resuilts uising solulble enzyme preparationis from chloroplasts of bean and( spiniach leaves (10, 11, 13). However, nonie of them has testedI the coupled enizyme system of pyrophosphorylase and(I trans- glulcosylase. This couipling reaction system is in- (leed considered to be essential to (lemonstrate the role of ADP-gluicose as aln intermediate in the starch synthesis. ?Mlurata and -Akazaw a (21 ) have specullated that photosynthetically prodl.iced ATP cotld]l be readily uitilized for the starch synthesis by transforming glicose-l-P, one of the iniitial photo- synthetic suigar , to ADP-glucose. Re- stilts showin in table I suipport this presumption. The reactioin system conisistinig of both ADP-glui- FRACTION NUMBER cose-pyrophosphorylase (soluible enzzyme) and(-ADP- FIw. 4. IoII exchlange colunin ch1ronmatographic separa- gluicose-starch transglucosylase (chloroplast starch tion of sucrose-P. Reaction mixture contained (in grantules) was the only efficielnt olne for starch ,umoles); tris buffer (pH 7.4), 400; NaF, 400; MgSO4, synthesis (Expt. II-Iv), notwithstanding that the 9.0; UDP-glucose-14C, 2.2 (1.9 X 10-2 ,uc, 4.7 X 10-4 rate of ADP-gluicose formatioln was lower than cp1m); fructose-6-P, 100; soluible enzyme preparation of that of UIDP-gliucose. However, glycerate- -P, rice leaves, 200 IAl in a total volume of 1.26 ml. Reaction P-enolpyruivate and(I fruLctose-1, 6-B, (lidl Inot exhibit NN as continued for 60 minuites at 370. Afterwards, de- ionized reactanit Nx as deioiiized and applied to a column ainy stimulating effect for the gluicose tranisfer from of Dowvex-1 X 8 (Cl- form) (0.8 X 7.0 cm) ; eluted glucose- -P via ADP-gluicose. Recent eleganit stuidy by the stepwise metlho(d as indicated in the figure. of Ghosh and Preiss (13) has shown the stimutla- Table I. Incor-por-ation of GljcosC 14C into Starch Concenitration of substrates contained in reaction mixture was (in ,umoles): ADP-glucose-14C, 0.185 (2.7 X 104 cp)III); UDP-glucose-'4C, 0.16 (1.8 X 104 cpm) ; glucose-14C-1-P, 0.5 (5.9 X 104 cpm); and either ATP, 0.5 or UTP, 0.5. Abbrevimtions used wvere: 3PGA, 3-phosphoglycerate; PEP, P-enolpyruivate; anid FDP, fructose-1,6-diP. Incubation Radio- % Ilucose-'4G time activitv incorporation Elxpt. Glucose (lonior system Enzvnme souirce nMil Cplln "into ~-thIrc7h IADP-glucose-'14C Grianutiles of clhloroplast starch (5.0 mg) 60 19,500 7.7 UDP-glucose-14C ,, 580 .) .3t Gluicose-' 4C-1 -P+ATP Granules of chloroplast starch (5.0 mg) 60 6200 1).n II + soluble enzyme (50 ,ul) Glucose-' 4C-1 -P+UTP ,. ..I 965 1.6, Glucose-' 4C-1-P+ ATP Grainulles of chloroplast starch (5.0 mg) 30) 8000 13.6 + soluible enzyme (50 ul) ,. ..~ 60 10,700 18.1 lIt 1 20 11,600 19.7 " "1 +3PGA(2.5mnr\i) 30 7450 12.6 ,. .. .. 60 9800 16.o6 120 11,900 20.2 Gluicose-l4C-1-P+ATP Granules of clhloroplast starch (5.0 mig) 6(0 80( 13.6 + soluible enzyme (50,l) " " +3PGA(m1-m) ,. . 15 9300 15.8 I 1 " (2.5mM) 98%00 16.6 IV ((5.OmM) 8900 15.1 " "-+PEP (2.5mnMI ) "~ 9600 16.3 "+FDP(2.5mnro) *' 11,400 IQ.3 NOMURA ET AL.-CHLOROPLAST STARCH .331 tionl of highl) purified (260 fold) ADP-glucose- 2. BASSHAM, J. A. 1965. : The patlh of pyrophosphorylase of spinach leaf by 3-phospho- carbon. In: Plant . J. Bonner and glycerate and other glvcolytic intermediates and J. Varner, eds. Academic Press, New York. p inferred the important regulatory role of the 875-902. 3. BIRD, I. F., H. K. PORTER, AND C. R. STOCKING. enzyme in the starch synthesis of leaf tissues. 1965. Intracellular localization of enzvme associ- Reason of our failure to reconfirming their restults ated wN-ith sucrose synthesis in leaves. Biochim. will certainly be due to the use of crude enzyme Biophys. Acta 100: 366-75. in our system. It is our opinion that more thorough 4. BUCHANAN, J. G. 1953. The path of carbon in experimentation is needed to prove whether or not photosynthesis. XIX. The identification of su- the point of control in the storage crose phosphate in sugar beet leaves. Arch. Bio- biosynthesis in plants is, unlike the case in the chem. Biophys. 44: 140-49. , at the level of ADP-glucose-pyrophos- 5. BUCHANAN, J. G., J. G. BASSHA-I, A. A. BENSON, phorylase, instead of the D. F. BRADLEY, M. CALVIN, L. L. DANS, M. GOOD- transglucosylase. It MAN, P. 'M. HAYES, V. H. LYNCH, L. T. NORRIS, should be pointed out in this regard that ADP- AND A. T. WILSON. 1952. The path of carbon in gluicose is in fact utilized for the formation of photosy-nthesis XVII. Phosphorous comlpound as suicrose (cf. fig 3). intermediates in photosynthesis in phosphorous me- It is interesting to recall that before the ctur- tabolism. W. D. 'McElroy and B. Glass, eds. Johns rently prevailing concept of the starch biosynthesis Hopkins University Press, Baltimore. p 440-66. 6. BURMA, D. P. AND D. C. MORTIIMER. 1956. The through the ADP-glucose pathway is established by biosyinthesis of uridine diplhosphate glucose anid the original work of Recondo and Leloir (25) in sucrose in sugar beet leaf. Arch. Biochemii. Bio- 1961, showing the mtuch faster svnthesis of starch phys. 62: 16-28. from ADP-glucose than that from UDP-glucose, 7. CALVIN, MI. AND J. D. BASSHA-M. 1962. The photo- radioisotopic experiment of Katuss and Kandler synthesis of carbon compounds. W. A. Benjamini, (17) had shown the close correspondence of the Inc., 'New York. ADP-glucose labelling and starch formation in 8. CARDINI, C. E. AND E. RECONDO. 1962. Specificity photosynthesizing Chlorella cells. However, in the of nucleoside diphosplhate in sucrose biosyn- general thesis. Plant Physiol. 3: 313-18. scheme of starch biosynthesis in reserve 9. DUBOIS, Y., K. A. GILLES, J. K. HAMIILTON- P. A. organs, suich as and , the interaction of REBEIRS, AND F. S-MITH. 1956. Colorimiietric UDP-gluicose and ADP-glucose has been fouind to method for determiiniation of sugars anid related be rather complex, and their control mechanism substances. Anal. Chem. 28: 350J-56. remains to be stutdied (1). In fact, in agreement 10. FRYD-MAN, R. B. AND C. E. CARDINI. 1964. Sol- with a view presented by Leloir (18), we cannot ub!e related to starclh sinthlesis. Biochem. ignore completely the UDP-glucose pathway of Biophys. Res. Comniilil. 17: 407-11. starch synthesis in rice seeds (23). Outr 11. Giiosri, H. P. AND J. PREISS. 1965. The biosyn- present experiments, however, have shown the thesis of starclh in spiinaclh chloroplasts. J. Biol. specific, predominant role of ADP-glutcose in starch Chem. 240: 960-62. synthesis of chloroplasts, whereas both ADP-glut- 12. GHOSii, H. P. AND J. PREISS. 1965. Biosyn1thesis cose and UDP-glutcose are synthesized by the re- of starch in spinaclh chlorop!asts. Biochemiistry 4: spective pyrophosphorylase sy-stem residing in 1354-61. chloroplasts. Hence, we feel that the utniqtue fea- 13. GHOSH, H. P. AND J. PREISS. 1966. Adeinosine diphosplhate glucose p rophosphorylase. A regu- tulre of the starch synthesizinig system associated latorv enzyme in the biosvynthesis of starch in with the chloroplast starch is distingulishable from spiniaclh leaf clhloroplasts. J. Biol. Chemii. 241: the one residing in starch granutles. It wotuld 4491-504. be of valuie to study the biophysical characteristics 14. HAQ, S. AND XNV. Z. HASSID. 1965. Biosyn1thesis of chloroplastic and reserve starch. of sucrose phosplhate with sugar cane leaf chloro- plasts. Planit Physiol. 40: 591-94. 15. HAWKER, J. S. AND 'M. D. HATCH. 1966. A spe- Acknowledgment cific sucrose phosplhatase from plant tissues. Bio- chem. J. 99: 102-07. This w-ork was initiated at the International Rice Re- 16. HEBER, U. AND J. search Institute when two of us, (TM and TA) were WILLEXBRINK. 1964. Sites of on duty- from 1962 to 1964. They record their deep ap- syvithesis anid transport of photosynthetic products preciation to the Institute officers for their support to within the leaf cell. Biochim. Biophys. Acta 82: this work. 313-14. 17. KAUSS, voN H. AND 0. KANDIER. 1962. Adeniosin triphosphat gltucose atis Chlorella. Z. Naturforsch. Literature Cited 17b: 588-860. 18. LELOIR, L. F. 1964. Nucleotide diphosplhate sugars 1. AKAZAWA, T. 1965. Starch, inulini and other re- anid saccharide syntlhesis. Biochem. J. 91: 1-8. serve . In: Plant Biochemistry. J. 19. MADISON, J. H., JR. 1956. The intracellular loca- Bonner and J. Varner, eds. Academic Press, New tion of phosphorylase in tobacco (Nicotiana taba- York. p 258-97. curni L.). Plant Phvsiol. 31: 387-92. 332 PLANT PHYSIOLOGY 20. MIENDICINO, J. 1960. Sucrose phosphate synthesis diphosplhate glucose anid starch synthesis. Bioclhem. in wlheat germ and greeni leaves. J. Biol. Chem. Biophys. Res. Commun. 6: 85-88. 12: 3347-52. 26. ROE, J. H. 1934. A colorimetric method for the 21. NIURATA, T. AND T. AZAKAN A. 1964. The role of determination of fructose in blood and urinie. J. adeniosinle diphosphate glucose in leaf starch for- Biol. Clhem. 107: 15-22. mationi. Biochem. Bioplhvs. Res. Commun. 16: 27. SHEN, L. AND J. PREISS. 1964. The activationi anid 6-11. inihibition of bacterial adenosine-diphosphoglucose 22. MURATA, T., T. SUGIYAMA, AND T. AKAZAWA. p)yrop)hosphorylase. Biochem. Bio>phys. Res. Coni- 1964. Ennzmicmi echianiism11 of starchl svynthesis in mun. 17: 424-29. 28. R. XI. 1963. of rice . II. Adenosine diplhosphate S-IL.LIE, Formiiationi anid funiction rip)enin1g soluble in glucose patlhva Arch. BiochIem. Biophvs. 107: proteiin clhloroplast. Can. J. Botanlv. 41: 123-54. 92- 101. 29. ST'OCKIN-G, C. R. 1952. The intracellular location 2). -MURATA, T., T. SUC;IYAAT.\, T. MINA-MIKAWVA, AND of phosphors lase in leaves. Anm. J. 39: T. AKAZA\\ A. 1966. Enz.ymic imieclhaniism of 283-87. starch1 in rice grains. III. synthesis ripening 30. STOCKING, C. R. 1959. Chloroplast isolationi in Mechanism of the sucrose-starclh conversion. Archi. media. Plant Physiol. 34: 56-61. Bioclhem. Biophvls. 113: 34-44. non-aqlueous 31. SToCKING, C. R., G. R. WILLIAMIS, AND A. ONGUN. 24. PREISS, J., L. SHEN, ANI) 'M. PARTRIDGE. 1965. 1963. Intracellular distributioni of the early prod- The activationi of Esch(iric)ita co/i ADP-glucose Ucts of photosynthesis. Bioclhemii. Riophys. Res. Ip ropliospiory lase. Biochlemii. Biophl s. Res. Coni- Commuln. 10: 416-21. m11un1. 18: 180-85. 32. STREET, H. E. 1963. Planit MIeta,bolismi. Per-a- 25. RfCON-DO, A-ND L. F. .LEiOIRm. 1961. Adenosin1e moni Press, Oxford.