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J. Gen. Appl. Microbiol., 15, 387-398 (1969)

STUDIES ON THE FERMENTATIVE PRODUCTIONF L-PROLINE

V. MECHANISM OF L-PROLINE PRODUCTION BYREVIBACTERIUM FBAVUM 2247 NO. 14-51

UMIHIRO YOSHINAGA

entral Research Laboratories of Ajinomoto Co., Inc.,

uzuki-cho, Kawasaki, Japan

Received October 30, 1968)

ddition of L-proline to the production medium for cultivation of straino. 14-5, an isoleucine auxotrophic mutant of Brevibacterium flavum 2247,

educed L-proline accumulation by about 40%. The formation, but not thectivity, of glutamate kinase, the first enzyme required for L-proline bio-

ynthesis from L-glutamate, seems to be controlled by L-proline. No dif- erence was observed in specific activities of this enzyme assayed in vitro

n either strain No. 14-5 or the parent strain.uring the growth of strain No. 14-5 (lacking in threonine dehydratase

ctivity), the increase of intracellular glycine, methionine, aspartate, andhreonine was considerably greater than in the parent strain. Intracellularaline, leucine, and lysine also increased. L-Proline produced was reducedo 42.9% without changing the level of intracellular glutamate by the ad-

ition of L-threonine and L-lysine to the medium before cultivation, but L- roline production was increased by nearly 20% when both amino acids

ere added near before the growth of this strain arrived at the stationaryhase. Glutamate kinase formation was repressed by nearly 20% by theddition of both amino acids, whereas the activity was not inhibited.

cell homogenate of the parent strain, which was not able to produce-proline in the medium, did produce L-proline from L-glutamate in theresence of ATP. From these findings, it was concluded that in strain No.4-5 both the higher level of available ATP resulting from the inhibitionf aspartate and homoserine kinase and the accumulation of intracellular-glutamate promoted by biotin-rich condition contributed to the abundantroduction of L-proline through stimulation of the glutamate kinase reaction.

n previous papers (1, 2), it was reported that an isoleucine auxotrophicmutant, Brevibacterium favum 2247 No. 14-5, produced a large amount of

This paper was presented at the 16th Symposium on Association of Amino Acid

and Nucleic Acid, October 2, 1967.

87

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388 YOSHINAGA VOL. 15

L-proline directly from sugar and inorganic nitrogen in the medium, and theconditions favorable for L-proline production by this strain were described.Evidence was provided for the phosphorylation of L-glutamate (glutamatekinase reaction) as an intermediate step in the biosynthesis of L-proline fromL-glutamate in this mutant (3, 4). The possibility that L-glutamate was con-verted into L-proline via L-ornithine after acetylation was excluded by thefinding that all L-ornithine-requiring mutants derived from this isoleucine-requiring mutant produced L-proline abundantly (4).

he present paper deals with the mechanism of L-proline production byan isoleucine-requiring mutant and with the elucidation of the relationship

between the mutation site of this strain and its L-proline productivity.

MATERIALS AND METHODS

icroorganism. Brevibacterium flavum 2247 No. 14-5 (ATCC No. 15940)was usually used throughout this work.

edia and cultivation. Depending on the purpose of studies, variousmedia, as described in the legends to tables and figures, were employed bymodifying the standard medium for L-proline production (4) which contains,per liter, 100 g glucose as starch hydrolyzate, 60 g (NH4)2504, 1 g KH2P04,8 g MgS04.7H20, 0.01 g FeS04.7H20, 0.01 g MnS04.4H20, 150 mg L-isoleucine,

1 ml Ajieki (solution of amino acid mixture obtained by the hydrolysis ofsoybean protein, total nitrogen 2.4%. Ajinomoto Co., Inc., Tokyo), 1 g D-tartaric acid, 450 ,gig biotin, 1 mg thiamine hydrochloride, and 50 g CaC03(sterilized separately). The pH was adjusted to 7.0 with KOH.

he bacterium was cultured as previously described (1).reparation of cell free solution. As described in previous papers (3, 4),

cell homogenates prepared by disrupting the cells with alumina followed bycentrifugation at 900 x g to remove alumina were employed exclusively fordetermining the formation of L-proline from L-glutamate. Cell-free extractsobtained by sonic oscillation of these homogenates followed by dialysis for 3to 5 hr against 0.01 M Tris-H2S04, pH 7.6, were usually used as the sourceof crude enzyme.

ssay of enzyme activities. Glutamate kinase activity was assayed byspectrophotometry, as previously described (3, 4), using the Hitachi Model139 UV-VIS Spectrophotometer. After incubation at 31 in a water bath,1.5 volumes of a mixture consisting of 10% FeCl3. 6H2O, 3.3% trichloroaceticacid, and 0.7 N HCl was added to the reaction mixture and the precipitateformed was removed by centrifugation. The optical density of the super-natant at 540 mp was used as a measure of the relative amount of hydro-xamate formed, after correction for the color value obtained in the absenceof L-glutamate. Aspartate kinase activity was determined according to themethod of BLACK and WRIGHT (5).

rotein was estimated by the method of LOWRY et al. (6), or of

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1969 Fermentative Production of L-Proline 389

WARBURG and CHRISTIAN as modified by LAYNE (7).xtraction of intracellular amino acids. As described by SHIIO et al.

(8), the cells were harvested by centrifugation, washed four times with 0.1 Mphosphate buffer (pH 7.5), and suspended in distilled water. When the cellswere not washed, the cells harvested were suspended directly in distilledwater. Intracellular amino acids were extracted by autoclaving the cell sus-pension at 120 for 30 min.

nalytical methods. Each analysis was carried out as previously des-cribed (1). Amino acids were analyzed by the microbioassay method (8, 9).

ESULTS

Relationship between glutamate kinase activity and production of L Proline bystrain No. 14-5

ffect of L-proline on the biosynthetic pathway of L-proline in strainNo. 14-5 was examined, especially with regard to its effect on glutamatekinase which was described in previous papers (3, 4).

a) Effect of exogenous L-proline on endogenous L-proline production byfermentation : As shown in Table 1, L-proline, produced directly from sugar,in the presence of added L-proline in a concentration of 0.5'3.0%, wasnearly 60% of that produced without exogenous L-Proline. Thus, it appearsthat this strain has acquired such a potent proline productivity through

able 1. Effect of exogenous L-proline on L-proline production by fermentation.asal medium : Glucose (as starch hydrolyzate) 100 g, (NH4)2S04 60g, KH~PO4

g, MgSO4.7H2O 5 g, FeSO4.7H2O 0.01 g, MnSO4.4H2O 0.01 g, D-tartaric acid 1 g,Ajieki'' 0 .5 ml, L-isoleucine 150 mg, biotin 350 rig, thiamine hydrochloride 500 pg.aCO3 50 g, per liter. Fermentation was carried out at 31 for 72 hr.

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mutation that it is able to produce L-proline in a good yield even in thepresence of substantial concentration of exogenous L-proline. However, noeffect of exogenous L-proline was observed when added at 24 hr, i. e., at thestationary phase of growth.

b) Effect of exogenous L-proline on the growth of strain No. 14-5: Inthe experiment shown in Table 1, the amount of L-proline produced wascalculated on the assumption that L-proline added would not be dissimi-lated by this strain. If this strain did dissimilate the added L-proline, itwould be expected that growth would be accelerated by the addition of L-proline. However, as shown in Table 2, its growth was not promoted bythe addition of L-proline. Moreover, from the fact that the amount of L-

proline produced in each case was about the same, i. e., about 2.0 g/liter,independent of the amount of L-proline added, it seemed reasonable to cal-culate the L-proline produced by subtracting the L-proline added from thetotal L-proline found in the medium.

c) Effect of L-proline on the formation of glutamate kinase : Theeffect of exogenous L-proline on its biosynthetic pathway, especially in re-lation to glutamate kinase, was studied in a cell-free system. As shown inTable 3, glutamate kinase formation appeared to be repressed by nearly 20%when this strain was cultured in the presence of 0.1% L-proline. This resultseems to be in accord with the results shown in Table 1, namely, that theamount of L-proline produced was decreased by nearly 40% by the addition

of L-proline to the medium.d) Effect of L-proline on the in vitro activity of glutamate kinase : The

addition of 20 mM L-proline had little or no effect on the activity of glutamatekinase (Table 4). This finding is also in accord with the results shown inTable 1, namely, that exogenous L-proline had no effect on endogenous L-proline production when added at 24 hr. Glutamate kinase activity from theparent strain was not inhibited either.

e) Comparison of activity of glutamate kinase in strain No. 14-5 and

able 2. Effect of L-proline added to the production medium before cultivationn the growth of strain No. 14-5.

ermentation was carried out at 31 for 24 hr employing the same medium asiven in Table 1.

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1969 Fermentative Production of L-Prohne 391

in parent strain 2247: Because of the increased proline production by themutant strain No. 14-5, glutamate kinase activities of both mutant and parentstrains were compared so as to determine whether this activity might beresponsible for the difference in L-proline production. No difference in thespecific activities of glutamate kinase was observed in the two strains (Table 5).

Table 3. Effect of L-proline on the formation of glutamate kinase.

ells were grown at 31 for 24 hr with or without L-proline (1 mg/ml) in thestandard medium.

he reaction mixture was incubated at 31 for 1.5 hr in a volume of 1.0 ml

containing 100 pmoles of L-glutamate (adjusted to pH 7.6 with KOH), 20 imoles ofATP, 20 imoles of MgSO4.7H2O, 400 pmoles of NH2OH, 80 ,moles of Tris-H2SO4

(pH 7.6), and 0.5 ml of dialyzed enzyme solutin (0.5 mg as protein).

Table 4. Effect of L-proline on the activity of glutamate kinase.

he reaction mixture was incubated at 31 for 1.5 hr in a volume of 1.0 mlcontaining 100 ,moles of L-glutamate (adjusted to pH 7.6 with KOH), 20 pmoles ofATP, 20 imoles of MgSO4.7H2O, 400 imoles of NH2OH, 80 pmoles of Tris-H2SO4

(pH 7.6), and 0.5 ml of dialyzed enzyme solution (0.50 mg as protein).

Table 5. Activity of glutamate kinase in strain No. 14-5 and parent strain 2247.

he parent strain 2247 was grown at 31 for 24 hr without isoleucine in thestandard medium.

he reaction mixture was incubated at 31 for 1.5 hr in a volume of 1.0 mlcontaining 100 imoles of L-glutamate (adjusted to pH 7.6 with KOH), 20 pmoles ofATP, 20 imoles of MgSO4. 7H2O, 400 j moles of NH2OH, 80 pmoles of Tris-H2SO4

(pH 7.6), and 0.5 ml of dialyzed enzyme solution (0.5 mg as protein).

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Relationship between the requirement for L-isoleucine and L proline production

train No. 14-5 has been found to lack threonine dehydratase activity(2), and a possible relation of the deficiency of this enzyme in L-glutamicacid-producing bacteria to the abundant production of L-proline has beenproposed (2). Accordingly, probable changes caused by this deficiency suchas an increase in intracellular L-threonine was examined.

a) Amino acid composition of the free internal pool in strain No. 14-5and parent strain 2247 grown in the L-proline production medium : The dataobtained on the intracellular composition of free amino acids in both strainNo. 14-5 and its parent strain are shown in Table 6. Glycine, methionine,aspartate, and threonine were obviously at a higher concentration in themutant form than in the parent strain, especially before washing. In addition,

Table 6. Amino acid composition of the free internal pool ofnd parent strain 2247 grown in the proline production

he parent strain 2247 was grown at 31 for 24 hr withoutstandard medium. A correction was made for contamination ofin cases where the cell paste was analyzed before washing four

phosphate buffer (10).

strain No. 14-5

medium.

isoleucine in the

external medium

times with 0.1 M

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the amino acids, valine, leucine and lysine, also increased, whereas alaninedecreased markedly. If glycine is assumed to be derived from threonine viathe threonine aldolase reaction, it is interesting that all of the intracellularamino acids (other than proline) which are strikingly increased are thosebelonging to the aspartic family.

b) Effect of L-threonine and L-lysine added to the medium :he striking increase of L-threonine suggested the possibility that the

accumulation of this amino acid might be related to the increase in prolineproduction by strain No. 14-5. Therefore, the effects of L-threonine and L-lysine, which have been reported (11) to be concerted feedback inhibitors ofaspartate kinase in the parent strain 2247, were examined in detail. It wasfound that L-proline produced was reduced to 42.9%, by the addition of L-threonine (0.6 g/liter) and L-lysine (3.0 g/liter as its hydrochloride) to themedium. As shown in Table 7, no change was observed, under the sameconditions, in the level of intracellular glutamate, a precursor of proline,which was not discharged as extracellular glutamate depending on the biotin-rich condition (8) just for the production of L-proline.

c) Effect of L-threonine and L-lysine added to the medium during cellgrowth on L-proline production : The accumulation of L-proline was increasedby nearly 20% when threonine and lysine were added to the productionmedium before the growth of these cells arrived at the stationary phase(Table 8). This result implies that the level of intracellular threonine playsan important role in the abundant production of L-proline, and that an in-crease in proline productivity in strain No. 14-5 could be related to an in-crease of intracellular threonine. In turn, this increase of threonine withthe growth of this microorganism depends on the deficiency of threoninedehydratase activity.

d) Effect of L-threonine and L-lysine on the formation of glutamatekinase : In the dialyzed cell-free extracts from the cells grown under theconditions described in Table 7, the specific activity of glutamate kinase wasrepressed by 22.1% (Table 9). This decrease of enzyme activity might besufficient to explain the decrease in L-proline accumulation caused by the

able 7. Effect of L-threonine, L-lysine, and L-methionine added to theedium on L-proline production by fermentation.

ermentation was carried out at 31 for 72 hr employing the standard medium.

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addition of both amino acids as shown in Tablee) Effect of L-threonine and L-lysine on

kinase and aspartate kinase : As demonstrated

7.

the activities

by the data in

of glutamate

Table 10, the

Table 8. Effect of L-threonine and L-lysine added to the medium during

ell growth on L-proline production by fermentation.

Fermentation was carried out at 31 for 72 hr employing the standard medium.

Table 9. Effect of L-threonine and L-lysine on the formation of glutamate kinase.

ells were grown at 31 for 24 hr with or without L-threonine (0.6 g/liter) andL-lysine HCl (3.0 g/liter) in the standard medium.

he reaction mixture was incubated at 31 for 1.5 hr in a volume of 2.0 ml

containing 200 ~imoles of L-glutamate (adjusted to pH 7.6 with KOH), 40 tmoles ofATP, 40 pmoles of MgSO4.7H2O, 800 ,moles of NH2OH, 80 pmoles of Tris-H2SO4(pH 7.6), and 1.0 ml of dialyzed enzyme solution (1.5 mg as protein).

Table 10. Effect of L-threonine and L-lysine on the activities of

lutamate kinase and aspartate kinase.

he reaction mixture was incubated at 31 for 1.5 hr in a volume of 2.0 mlcontaining 200 ,moles of L-glutamate or 400 pmoles of L-aspartate (adjusted to pH7.6 with KOH), 40 ,moles of ATP, 40 ,moles of MgSO4.7H2O, 800 ,moles of NH2OH,80 ,moles of Tris-H2SO4 (pH 7.6), and 1.0 ml of dialyzed enzyme solution (1.50 mgas protein).

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1969 Fermentativ e Production of L-Proline 395

addition of both amino acids had no effect on glutamate kinase activity,whereas the inhibition of aspartate kinase in this mutant as well as in theparent strain was confirmed.

f) Effect of ATP on proline formation by cell homogenate of parentstrain 2247: Referring to Fig. 1, the findings reported above lead to thefollowing proposals for the mechanism of L-proline production in strain No.14-5. In this strain, an increase of intracellular threonine (whose level wasvery low in parent strain 2247 (8)) caused by the deficiency in threoninedehydratase would bring about an inhibition of aspartate kinase. This in-hibition would be further increased by lysine, and would result in the spar-ing of ATP. In addition, the inhibition of homoserine kinase by threonine,which was observed in the parent strain (11), might be sufficient to spareATP as well. The higher level of available ATP resulting from the in-hibitions of both aspartate and homoserine kinase might act to more effectivelypromote the glutamate kinase reaction which requires ATP as a cofactor.Finally, intracellular L-glutamate, the accumulation of which was found tobe promoted by biotin-rich condition (8), would serve as a substrate for theproduction of L-proline in the medium. That ATP might indeed be limitingfor L-proline production in strain No. 14-5 seems to be indicated by the factthat a cell homogenate of the parent strain, which was not able to produceL-proline in the medium, did produce L-proline from L-glutamate in the pre-sence of ATP as shown in Table 11.

Fig.

bacterium

1. Relationship between

flavuin.

the glutamate and aspartate families in Bievi-

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DISCUSSION

t has been suggested by STRECKER 12), BAICH and PIERSON (13), and byTRISTRAM nd THURSTON 14) from experiments with intact cells of E, coli thatone of the early steps (probably the first step) in the biosynthetic pathway of L-proline from L-glutamate is subject to end-product inhibition, thus controllingL-proline biosynthesis. However in Brevibacterium flavum, glutamate kinase,

the first enzyme of L-proline biosynthesis, was not inhibited by L-proline.If the metabolic pathway for the reduction of L-glutamate to L-proline, whichwas observed in strain No. 14-5 (4), is not the same as the pathway foroxidation of L-proline to L-glutamate, as suggested by S TRECKER (15), bio-synthesis of L-proline is presumably controlled in this microorganism by somemechanism other than the feedback inhibition of glutamate kinase by L-pro-line. If the concentration of biotin in the medium is low in such a case,intracellular L-glutamate reduced from L-proline will be excreted as extra-cellular L-glutamate, but, at a higher concentration of biotin just for theproduction of L-proline, it is impossible that intracellular L-glutamate is dis-charged as extracellular L-glutamate itself or L-glutamine (the latter is known

to be produced very easily at high NH4+ concentration (16)). In other words,this fermentative production of L-proline might be regarded as a kind ofdetoxication of intracellular L-glutamate by converting less permeable L-glutamate into more permeable L-proline (4). BAICH and PIERSON (13) andTRISTRAM and THURSTON 14) proposed also that overall control of L-prolinebiosynthesis, besides involving end-product inhibition, also involved enzymerepression. In our experiments, glutamate kinase formation in Brevibacteriumflavum was repressed by nearly 20% when 0.1% L-proline was added to theculture medium.

urther interesting data were obtained by comparing the intracellularfree amino acid concentration in strain No. 14-5 with that of the parent

Table 11. Effect of ATP on the proline formation by the cellomogenate of parent strain 2247.

ells were grown at 31 for 24 hr without isoleucine in the standard medium.he reaction mixture was incubated at 31 for 5 hr in a volume of 3.0 ml con-

taining 100 pmoles of L-glutamate (adjusted to pH 7.6 with KOH), 60 pmoles ofMgSO4 7H2O, 300 pmoles of Tris-H2SO4 (pH 7.6), and 1.0 ml of cell homogenate

(37.0 mg as protein).

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strain. In addition to the expected increase of intracellular threonine result-ing from threonine dehydratase deficiency, the amount of aspartate andmethionine also increased markedly. The increase of aspartate is probablydue to inhibition of aspartate kinase by threonine and lysine, while the in-crease of methionine appears to be due to inhibition of homoserine kinaseby threonine (11). This proposed role for the relation of intracellular thre-onine to the abundant production of L-proline appears to be supported by thecharacteristic decrease of L-proline production resulting from such decreaseof intracellular threonine, as is obtained by increasing the concentration ofL-isoleucine, reported to be an inhibitor of homoserine dehydrogenase activity

(11), or by adding L-methionine to the medium which is known to represshomoserine kinase (17). Thus, it is concluded that ATP plays the most im-portant role in the fermentative production of L-proline in strain No. 14-5,although the effect of cofactors ether than ATP also has to be taken intoaccount. The proposed mechanism might be called fermentation of metaboliccontrol, reflecting the complexity of metabolic control in microorganisms.

n connection with this mechanism, it is interesting that an increase ofL-proline biosynthesis by ATP in plants also has been reported recently. Intobacco leaves formation of L-proline from L-glutamate was promoted bylight irradiation (18), and it was reported that this phenomenon was chieflydue to ATP synthesis caused by noncyclic photophosphorylation. No effect

of ATP or ADP was seen in young leaves with potent biosynthesis of L-proline but L-proline formation in old leaves with weak biosynthesis of L-proline was accelerated by the addition of either cofactor (19).

ecently, fermentative production of L-proline by histidine auxotrophicmutants of Brevibacterium flavum (20) and Brevibacterium sp. No. 7996 (21)has also been reported. L-Proline productivity of Brevibacterium sp. No. 7996YS-48 was said to be far better than its parent (22), but its mechanismremains unsolved. However, the leading role of ATP in L-proline productionindicated for an isoleucine auxotrophic mutant might perhaps be applicablealso to these histidine auxotrophic mutants in relation to the biosyntheticpathway of histidine.

he author is grateful to Dr. H. J. Strecker, Professor of Biochemistry, AlbertEinstein College of Medicine, Yeshiva University, for his valuable suggestions. Theauthor also wishes to thank Drs. T. Yoshida, T. Tsunoda, N. Katsuya and S. Okumuraof this laboratories for their interest and encouragement during the course of thepresent work. Much appreciation is expressed to Mr. Y. Takeda and Mrs. M. Sato fortheir technical assistance.

REFERENCES

) F. YOSHINAGA, . KONISHI, . OKUMURA nd N. KATSUYA, . Gen. Appl. Microbiol.,2, 219 (1966).

) F. YOSHINAGA, . YOSHIHARA, . OKUMURA nd N. KATSUYA, . Gen. Appl. Micro- iol., 13, 25 (1967).

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F. YOSHINAGA, Y. TAKEDA and S. OKUMURA, Biochem. Biophys. Res. Commun., 27,143 (1967).F. YOSHINAGA, Nippon Nogeikagaku Kaishi, 42, 703 (1968), in Japanese.S. BLACK and N.G. WRIGHT, J. Biol. Chem., 213, 39 (1955).OH. LOWRY, N.J. ROSEBROUGH, AL. FARR and R.J. RANDALL, J. Biol. Chem., 193,235 (1951).E. LAYNE, In Methods in Enzymology, 3, ed. by S.P. COLOWICK and NO. KAPLAN,Academic Press Inc., New York (1957), p. 447.I. SHIIO, K. NARUI, N. YAHABA and M. TAKAHASHI, J. Biochem. (Tokyo), 51, 109

(1962).T. TSUNODA, In Chemistry of Proteins, 1, ed. by S. Akabori and S. Mizushima,Kyoritsu Shuppan Co., Ltd. Tokyo (1954), p. 282.I. SHIIO, S. OTSUKA and M. TAKAHASHI, J. Biochem. (Tokyo), 51, 56 (1962).R. MIYAJIMA, S. OTSUKA and I. SHIIO, J Biochem. (Tokyo), 63, 139 (1968).H.J. STRECKER, J. Biol. Chem., 225, 825 (1957).A. BAICH and D.J. PIERSON, Biochim. Biophys. Acta, 104, 397 (1965).H. TRISTRAM and C.F. THURSTON, Nature, 212, 74 (1966).H.J. STRECKER, J. Biol. Chem., 235, 3218 (1960).K. OSHIMA, K. TANAKA and S. KINOSHITA, Amino Acids, 7, 73 (1963), in Japanese.R. MIYAJIMA, S. OTSUKA and I. SHIIO, Proc. Ann. Meet. Agr. Chem. Soc. Japan, p.272 (1968), abstract in Japanese.S. MIZUSAKI, M. NoGucHI and E. TAMAKI, Arch. Biochem. Biophys., 105, 599 (1964).M. NOGUCHI, A. KOIWAI, M. YOKOYAMA nd E. TAMAKI, Plant & Cell Physiol., 9,35 (1968).

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