BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96016

ENHANCEMENT BY 2,4-DICHLOROPHENOXYACETIC ACID OF

CHROMATIN RNA POLYMERASE IN SOYBEAN HYPOCOTYL TISSUE

35

T. J. O'13RIEN, B. C. JARVIS, JOE H. CHERRY AND J, B. HANSON

Horticulture Department, Purdue University, La]ayette, Ind. and Agronomy Department, University o] Illinois, Urbana, Ill. (U.S.A.)

(Received May 2oth, 1968)

SUMMARY

Chromatin isolated from control and 2,4-dichlorophenoxyacetic acid-treated soybean hypocotyl tissue incorporates labeled nucleoside triphosphates into acid- insoluble RNA. Chromatin RNA polymerase activity of soybeans depends on the presence of all 4 nucleoside triphosphates, Mg *+ and Mn 2+. RNA synthesis is inhibited by pyrophosphate and actinomycin D. The product of the reaction is completely sensitive to ribonuclease. Chromatin preparations from 2,4-dichlorophenoxyacetic acid-treated plants have a higher polymerase activity than control chromatin. RNA polymerase of chromatin preparations shows no response to 2,4-dichlorophenoxy- acetic acid added during incubation. RNA polymerase from Escherichia coli greatly augments RNA synthesis of chromatin preparations from control and 2,4-dichloro- phenoxyacetic acid-treated plants, but both chromatin preparations exhibit similar saturation curves with added E. coli polymerase. Comparison of the RNA products of chromatin preparations from control and 2,4-dichlorophenoxyacetic acid-treated plants on methylated albumin kieselguhr columns reveals a change in the RNA product.

INTRODUCTION

The nucleic acid content of a number of plant tissues is greatly increased as a result of t reatment with one of a variety of different hormones. Notably, auxins enhance RNA synthesis in intact plants 1,2, excised tissue ~,4 and isolated nuclei preparations 5-7. The gibberellins augment nucleic acid synthesis in barley seedlings s and isolated nuclei 9. The kinins, a third group of substances with hormone activity, also appear to modify nucleic acid synthesis 1°. While these 3 groups of plant hormones appear to directly or indirectly mediate a regulatory control through nucleic acids, little information is available on the mechanisms of hormone-stimulation of nucleic acid synthesis in plants. In the case of intact tissues, hormone treatment might result in an increased nucleic acid content if the rate of synthesis is enhanced or the rate of degradation decreased. Previous experiments on nuclei preparations from soybean hypocotyls ix show that the auxin, 2,4-dichlorophenoxyacetic acid, stimulates the

Biochim. Biophys. Aeta, 169 (1968) 35-43

36 T.J. O'BRIEN et al.

incorporation of labeled precursors into nucleic acids by as much as two-fold. From these and other experiments we have considered the major affect of auxin on nucleic acid accumulation to be on the rate of nucleic acid synthesis. The rate of nucleic acid synthesis by isolated nuclei might be augmented by an increase in the endogenous enzyme complement or, in the case where DNA template is limiting, by making available more template.

This report deals with the relative contributions of RN A polymerase activity and template availability of chromatin isolated from hypocotyls of control and 2,4- dichlorophenoxyacetic acid-treated soybean plants. Since the chromatin from this tissue appears to contain most of the RNA polymerase activity, an assessment of the change in polymerase activity due to auxin t reatment is possible.

MATERIALS AND METHODS

Chromatin isolation. Soybeans /Glycine max L. Var. Wayne) were grown in vermiculite at 27 ° in the dark for 4 days prior to spraying with 2,4-dichlorophenoxy- acetic acid. Hypocotyl sections (taken I cm below the cotyledonary hook to the first lateral root) were excised at various times after t reatment and cooled on ice. Sub- sequent steps in the isolation procedure were performed at 3-4 °. IOO g lots of hypoco- tyls from 2,4-dichlorophenoxyacetie acid-treated and untreated plants were extracted according to the method of HUANG AND BONNER TM and the chromatin pellets suspended in 2 ml of 0.6 M Tris-HC1 (pH 8.o), and o.oi M Cleland's reagent. Aliquots of this suspension were used to assay chromatin RNA polymerase.

Chromatin RNA polymerase. The incorporation of labeled nucleoside triphos- phate into trichloroacetic acid-insoluble material was used as a measure of RNA synthesis. The reaction mixture contained in/~moles the following: GTP, o.2; CTP, o.2; ATP, 0.2; MgC12, I; ~nC12, 0.25; Cleland's reagent, I; Tris-HC1, (pH 8.o), 20 and IO #C of E3HJUTP (2.3 raC/~mole) and chromatin (equivalent to 3-6 #g of DNA) in a final volume of 0. 4 ml. The reaction was carried out at 37 ° for 15 rnin and stopped by the addition of 4 ml of io °/o ice-cold trichloroacetic acid. The precipitates were transferred to membrane filters (Schleicher and Schuell, type B-6) and washed with 25 ml of cold 5 % trichloroacetic acid. Filters were dried under infra-red lamps and counted in a Tri-Carb liquid scintillation spectrometer.

Methylated albumin kieselguhr column chromalography. The RNA product of the RNA polymerase reaction (5 × normal reaction) was fractionated on methylated albumin kieselguhr columns. The reaction was stopped by cooling the incubation mixture to 0-2 °. The labeled product was divided into 2 fractions, that bound or associated with the chroraatin DNA template and that which was unbound and confined to the supernatant after centrifugation at 20 ooo ×g for IO rain. Both RNA fractions were collected and prepared for methylated albumin kieselguhr column chromatography as described in Fig. 3.

Biochirn. Biophys. Acta, I69 (~968) 35-43

C H R O M A T I N R N A P O L Y M E R A S E 3 7

Chromatin R N A directed amino acid incorporation. The capacity of the chromatin RNA products to support amino acid incorporation was assayed in an in vitro system utilizing ribosomes from dry peanut seed which have little or no capacity for protein synthesis without added RNA (ref. 13). E14C~Leucine incorporation was determined with time by the filter disc technique 14.

DNA and protein determination. Chromatin-DNA was determined according to BURTON 15 after hydrolysis in 0.5 M perchloric acid at 7 °0 for 45 min. Protein was assayed by the method of LOWRY et al. lB.

R E S U L T S

The incorporation of [~H]UTP into RNA by chromatin RNA polymerase in the absence of exogenous DNA is completely dependent on the presence of ATP and CTP. The omission of GTP inhibits the reaction by 74 % (Table I). Omission of both

T A B L E I

REQUIREMENTS OF SOYBEAN HYPOCOTYL CHROMATIN R N A POLYMERASE

T h e c o m p l e t e r e a c t i o n m i x t u r e is d e s c r i b e d i n MATERIALS AND METHODS,

System pmoles [3H]UMP incorporated % inhibition per Ioo i~g DNA

C o m p l e t e 218 o - - M g ~+ 95 46 - - M n 2+ 143 35 - - M g 2+, M n 2+ 13 9 4 - - A T P 5 98 - - G T P 31 74 - - C T P 15 93 - - C T P , G T P , A T P 9 9 6

Mg ~+ and Mn ~+ inhibited the chromatin reaction by 94 % while the omission of only I cation inhibited by about 35 % to 45 %- It should be noted that Mg ~+ concentra- tions greater than 2.5 mM and Mn 2+ greater than 0.63 raM, whether in combination or singly, inhibit the reaction.

Pyrophosphate, one of the products of the reaction, drastically inhibits RNA synthesis while orthophospbate has little effect (TaNe I I). The addition of spermidine phosphate markedly inhibits chromatin-directed RNA synthesis which is in contrast with the results on the soluble RNA polymerase system from maize xT. While ammonium sulfate enhances the activity of chromatin preparations from mammalian tissue TM, it has no effect on the soybean chromatin system. The addition of ribonuclease to the reaction tube completely inhibits the incorporation of the E3H]UTP label into RNA, presumably by degradation of the product produced during the reaction.

Fig. I illustrates the kinetics of the reaction and shows maximum [3H]UTP incorporation within IO rain. The induction of chromatin-directed RN'A synthesis as

Biochim. Biophys. Acta, 169 (1968) 3 5 - 4 3

38 T, J, O'BRIEN e~ al.

TABLE II

E F F E C T S O F V A R I O U S A D D I T I V E S O N C H R O M A T I N ~ l ~ A P O L Y M E R A S E A C T I V I T Y

2,4-Dichlorophenoxyacetic acid t reatment of iooo /zg/ml was applied I2 h prior to isolation of chromatin.

pmoles [*H]UMP incorporated per zoo I~g DNA

Control 2,4-Dichlorophenoxyacetic acid

Complete 142 +Pyrophospha te (5/~moles) o

+Phospha te (5/*moles) 143 +Spermidine phosphate (5 #moles) 68 +Act . D (o.oi #g) 128 +Act . D (o.I/zg) 55 +Ac t . D (2.o/zg) 6 + Ribonuclease (IOO/zg) 5 + (NH4)6SO4 (2o #moles) I54 +2,4-Dichlorophenoxyacetic acid (lO -6 M) 139 +2,4-Dichlorophenoxyacetic acid (IO -6 M) 141

341 9

318 164 274 13o

2 2

9 336 339 345

a

2 5 0 -

o. 200-

,.q V-

O 150-

o z IO0-

%n 50- ---"- ' - ' -x ~

2,4-DICHLORODHENOXYACETIC ACID

o ~ / CONTROL

/ X ~

a

O0 1500

o.

g I 0 0 0

s g O Z

500 D

g

E ~x

~,4- DICHLOROPH ENOXY ACETIC ACiD

T I M E ( m i n ) UNITS OF ADDED E. coil RNA POLYMERASE

Fig. I. The kinetics of RNA synthesis directed by chromatin, and its associated RNA polymerase, extracted from the hypocotyls of control and 2,4-dichlorophenoxyacetic acid-treated soybean seedlings. Seedlings were sprayed with 2,4-dichlorophenoxyacetic acid (iooo/~g/ml) 12 h prior to chromatin isolation.

Fig. 2. Saturation of chromatin DNA from control and 2,4-dichlorophenoxyacetic acid-treated soybean seedlings with exogenous E. coli RNA polymerase. Chromatin DNA (o.3-o. 4/~g DNA) and its associated RNA polymerase from control and 2,4-dichlorophenoxyacetic acid-treated tissue was first assayed to determine the endogenous RNA polymerase activity. These levels of activity are plotted as the initial points on the Y axis of the saturation curves. In separate reaction mixtures E. cull RNA polymerase (I, 3 and 5 units respectively) was added to obtain tile remaining points on the saturation curve. Units of E. cull polymerase are those described by Biopolymers Inc. Pinebrook, New Jersey.

a function of 2,4-dichlorophenoxyacetic acid concentration is shown in Table I I I . The enhancement of chromatin RNA polymerase activity was not manifested whithin 2 h of 2,4-dichlorophenoxyacetic acid treatment (Table IV). However after 4 h and the ensuing duration of 2,4-dichlorophenoxyacetic acid treatment a continuous

Biochim. Biophys. Acts, 169 (1968) 35-43

C H R O M A T I N RNA P O L Y M E R A S E 3 9

T A B L E I I I

ENHANCED CHROMATIN ACTIVITY 12 h AFTER TREATMENT WITH A RANGE OF 2,4-DICHLOROPHENOXY- ACtgTIC ACID CONCENTRATIONS

Treatment i~g pmoles [SH] U M P incorporated Increase over 2,4-dichlorophenoxyacetic acid/ml per Ioo tzg DNA control (%)

O 113 .5 O IO I67 . 7 48 5 ° 284. I 151

IOO 3 5 7 . 5 215 IOOO 379.O 235

T A B L E I V

THE INDUCTION OF CHROMATIN DIRECTED R N A SYNTHESIS OVER A 24 h PERIOD

C h r o m a t i n w a s i s o l a t e d a t v a r i o u s t i m e s a f t e r 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d t r e a t m e n t ( i o o / 2 g / i n l ) , a s d e s c r i b e d i n MATERIALS AND METHODS.

Hours after pmoles [aHJ U M P incorporated Increase over treatment per xoo I*g DNA control (%)

o lO6 o 2 I i O 4 4 155 4 6 8 235 114

12 3 8 6 235 2 4 441 3Ol

increase in activity was observed. Actinomycin D inhibited chromatin preparations from control and 2,4-dichlorophenoxyacetic acid-treated plants equally well (Table II).

When chromatin preparations from control and 2,4-dichlorophenoxyacetic acid-treated plants were saturated with Escherichia coli RNA polymerase a greatly magnified RNA synthesis was observed (Table V). The endogenous chromatin RNA polymerase from control tissue contributed only 7 % of the total possible RNA

T A B L E V

POTENTIAL R N A SYNTHESIS OF CHROMATIN D N A FROM CONTROL AND 2,4-DICHLOROPHENOXYACETIC ACID TREATED PLANTS*

System pmoles [3H] U M P incorporated per 2oo tzg DNA

Control 2,4-Dichlorophenoxyacetic acid

E n d o g e n o u s R N A p o l y m e r a s e 117 S a t u r a t i o n w i t h E. coli R N A p o l y m e r a s e 1318 % P o t e n t i a l R N A s y n t h e s i s e x p r e s s e d b y e n d o g e n o u s R N A p o l y m e r a s e 7

243

I 4 8 3

16

P l a n t s s p r a y e d w i t h i o o o / * g / m l 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d 12 h p r i o r t o h a r v e s t i n g .

Biochim. Biophys. Acta, 169 ( I 9 6 8 ) 3 5 - 4 3

4 ° T.J. O'BRIEN et al.

synthesis. The latter was measured by saturation with E. coli RNA polymerase. 2,4-Dichlorophenoxyacetic acid chromatin RNA polymerase in comparison contrib- uted approx. 16 °/o of the total possible RNA synthesis. Both chromatin prepara- tions exhibited similar saturation curves and the rate of RNA production was maximum at 3 units of E. coli RNA polymerase per incubation (Fig. 2).

Charaderization o/ chromatin RNA produd. Methylated albumin kieselguhr column chromatography of the in vitro synthesized RNA products indicated differences between the product synthesized in the control system and that synthesized in the 2,4-dichlorophenoxyacetic acid system. The unbound RNA, that remained in the supernatant after centrifuging the chromatin from the cooled reaction mixture, was essentially spread over the whole elution profile (Fig. 3A and B). The unbound RNA

.oo I 350

3 0 0 i

250

200

t 5 0

O t 0 0

5O

A. CONTROL UNBOUND RNA

NH40H . ~ . ~,\

2'o ~o 4o do ,60 ,~o ,~o ,~o ,~o z'oo TUBE NUMBER

526

4"00~ I R 2 ,4_DiCHLOROPHENOXYACETIC ACID

350] UNBOUND RNA

300

2 2 3 3 250

2

150

0 I00 1.5M

50 NH40H

20 40 60 80 I00 t 20 140 ~60 180 200 TUBE NUMBER

1600- C CONTROL BOUND RNA

I '~ O0"

VJ 1200.

Z

800-

~- 600- g o ¢~ 40O-

t.SM 200- N;40 H

o 6o ,60 ~o ,~o ,~o ,go 2bo TUBE NUMBER

1600-

1400-

~ 120o-

$000.

800-

600-

0 400.

200-

D. 2,4-D~CNLOROPNENOXYACETtC ACID BOUND RNA

N H40H

iF~o ,o 60 ;o ,;o :,o ,~o ,80 2~o TUBE NUMBER

Fig. 3. Methylated a lbumin kieselguhr (MAK) column ch roma tog raphy of the RNA produc t synthesized by chromat in RNA polymerase. The p roduc t of the react ion was most ly dissociated from the chromat in (unbound RNA), a l though a small quan t i t y of RNA remained associated wi th the ch romat in (bound RNA) when it was centrifuged from the react ion mix ture at 2o ooo × g for io min. The chromat in -bound produc t was removed by phenol extract ion 19. Both the unbound and the bound RNA fract ions of the chromat in react ion were dialyzed overnight at 3 ° against o.o 5 M phospha te buffer (pH 6.7), pr ior to f ract ionat ion on methyla ted a lbumin kieselguhr columns 2°. Frac t iona t ion of the RNA was achieved by elution of the co lumn wi th a linear salt gradient (o.35 M N a C I - I . I 5 M NaC1 in o.o 5 M phospha te buffer, p H 6.7). Tenaciously bound RNA (ref. 21) which is no t eluted wi th a salt gradient was removed from the column with 1. 5 M NH4OH and immedia te ly neutralized. 5 ml fract ions were collected and the RNA precipi tated wi th io % trichloroacetic acid in the presence of i o o F g DNA. Precipitates were collected on membrane filters and counted as described above. Elut ion profiles of unbound RNA synthesized by chromat in prepara t ions f rom control and 2,4-dichlorophenoxyacetic acid-treated soybean hypocoty ls (12 h, zooo/~g/ml) are shown in A and B, respectively. Profiles of the bound RNA of the chromat in react ion are likewise shown in C and D.

Biochim. l~iophys. Acta, 169 (1968) 35-43

CHROMATIN R N A P O L Y M E R A S E 41

from the 2,4-dichlorophenoxyacetic acid reaction mixture did however show enhance- ment in several areas of the profile viz. in tube numbers 20-60, lOO-14o and in the tenaciously bound RNA fraction (Fig. 3B). The bound RNA products on the other hand (Fig. 3C and D) contained only I fraction of RNA, that which eluted in tubes 25-50 where 4-S and 5-S RNA are normally eluted under similar conditions. A notable increase in the bound RNA isolated from the 2,4-dichlorophenoxyacetic reaction mixture was also observed (Fig. 3C and D).

The RNA products synthesized in vitro by both control and 2,4-dichlorophen- oxyacetic acid chromatin had activity when assayed in amino acid incorporating system (Fig. 4). The product synthesized by chromatin from 2,4-dichlorophenoxy- acetic acid-treated plants was more effective in supporting amino acid incorporation than that of control chromatin. However, it should be noted that the 2,4-dichloro- phenoxyacetic acid reaction mixture normally resulted in 2 to 3 times more RNA pzoduct than that of the control. If the RNA polymerase reaction was carried out at 0-2 ° then very little activity was observed when the products were coupled to the amino acid incorporating system (Fig. 4).

O Z

(~ j 30 ' O ( t

t ~ t O Z O

~ ao . j :3.

~ e : 10

2,4- DrCHLOROPHENOXYACETIC ACID

0

CONTROL

o / ° l ° -

IS 30 4 5 6 0 TIME ( r n i n )

Fig. 4. The capac i t y of R N A s y n t h e s i z e d by endogenous R N A p o l y m e r a s e assoc ia ted wi th cont ro l a n d 2 ,4 -d ich lo rophenoxyace t i c acid c h r o m a t i n to s u p p o r t amino acid incorpora t ion as m e a s u r e d by s u b s e q u e n t El*C]leucine inco rpora t ion in to prote in . C h r o m a t i n r eac t ions (2 × normal ) of con t ro l a n d 2 ,4 -d ich lo rophenoxyace t i c ac id - t r ea ted p l a n t s were carr ied o u t a t 37 °. The R N A p o l y m e r a s e r eac t ion was s t opped b y cool ing in ice. A 0.5 ml a l i quo t of t he r eac t ion m i x t u r e was t h e n t r ans fe r r ed to t h e ami no acid i nco rpo ra t i ng s y s t e m 13 and a s s a y e d a t 37 ° for i t s c apac i t y to s u p p o r t a m i n o acid inco rpora t ion ( 0 - 0 , × - x ). E n d o g e n o u s R N A p o l y m e r a s e reac t ions carr ied o u t a t o -2 ° were t e s t ed in a s imi la r f a sh ion ( O - - - 0 , × - - - × ).

DISCUSSION

The enhancement of chromatin-directed RNA synthesis by 2,4-dichloro- phenoxyacetic acid provides a good basis for the previously noted accumulation of RNA in response to auxins and auxin-like herbicides 1-4. The change occurs in 4 h or less and might explain the enhanced RNA synthesis in vivo. A number of investi- gators have pointed out a differential effect of actinomycin D on DNA directed RNA synthesis ~2,23. In such cases low concentrations of actinomycin D preferentially inhibit nucleolar or ribosomal-like RNA synthesis. In a similar experiment with

B i o c h i m . B i o p h y s . A c t a , 169 (1968) 35-43

4 2 T . J . O'BRIEN et al.

soybean chromatin no such preferential effect was observed suggesting that the 2,4-dichlorophenoxyacetic acid-induced RNA synthesis is not largely ribosomal-like RNA.

The increased capacity of chromatin to synthesize RNA as a result of 2,4- dichlorophenoxyacetic acid-treatment can be ascribed to an increase in the amount of available DNA template or to an induction or activation of chromatin RNA poly- merase. We have been unsuccessful in isolating RNA polymerase from chromatin and therefore a direct estimate of RNA polymerase per unit of chromatin DNA is not possible. However, by employing E. coli RNA polymerase to saturate the chro- matin system an estimate of chromatin template efficiency can be made. The E. coli RNA polymerase saturation data suggest that 2,4-dichlorophenoxyacetic acid- t reatment does not change DNA template availability within a 12 h period. On this basis, it is concluded that the enhanced synthesis of RNA by 2,4-dichlorophenoxy- acetic acid-treatment results from a larger quant i ty of RNA polymerase associated to chromatin DNA. These DNA sites are apparent ly already available but are trans- cribed only as a consequence of 2,4-dichlorophenoxyacetic acid treatment.

The major conclusion concerning the influence of auxin on ~RNA synthesis clearly implicates RNA polymerase as the agent responsible for the enhanced RNA production in response to 2,4-dichlorophenoxyacetic acid-treatment. While 4 h are required to observe an increase in chromatin-directed RNA synthesis, the results are consistent with the idea that an increased level of RNA polymerase in auxin-treated tissue precedes an increased rate of RNA synthesis and subsequently more RNA.

As a working hypothesis we propose that auxin, 2,4-dichlorophenoxyacetic acid in our experiments, is an effector or co-effector substance and it reacts with the chromatin material which then leads to the production of more RNA polymerase. The increased quanti ty of enzyme becomes associated with the chromatin DNA and transcribes the previously available DNA template sites. Thus, in the presence of sub-optimal concentrations of auxin little RNA polymerase will be produced which would lead to little RNA synthesis and subsequently little protein production. In this case, control of any process which does not require RNA synthesis would not be affected by auxin. In order for this hypothesis to be proven at least partially correct, we need to show that 2,4-dichlorophenoxyacetic acid induces the synthesis of RNA polymerase.

The apparent availability of many DNA sites which are not transcribed by endogenous lZNA polymerase, but are transcribed by E. coli RNA polymerase, suggests specificity between endogenous RNA polymerase and certain DNA sites. One explanation might be that gene sites necessary for normal metabolic maintenance in the cell are at all times available, their transcription being dependent on the presence of RNA polymerase at these sites. These template sites might have different affinities for RNA polymerase and in cases where little RNA polymerase is available only sites having a high affinity for the enzyme would be transcribed. On the other hand, there may be several RNA polymerases each having different specificities.

ACKNOWLEDGEMENTS

This work was supported by a contract from the U.S. Department of Agri- culture (616-15-3) and a grant from the National Science Foundation (GB-4593) to

Biochim. Biophys. dcta, 169 (1968) 35-43

CHROMATIN R N A POLYMERASE 43

J . H . C . , a n d a g r a n t f r o m I n t e r n a t i o n a l M i n e r a l s a n d C h e m i c a l Corp. t o J . B . H . S o m e

of t h e r e s u l t s p r e s e n t e d in t h i s p a p e r we re i n c l u d e d in a t h e s i s b y O 'BRIEN a n d

s u b m i t t e d t o t h e G r a d u a t e School , U n i v e r s i t y of I l l i no i s in p a r t i a l f u l f i l l m e n t for

t h e P h . D . degree . Al so s o m e r e s u l t s of t h i s p a p e r we re p r e s e n t e d t o t h e S i x t h I n t e r -

n a t i o n a l C o n f e r e n c e o n P l a n t G r o w t h S u b s t a n c e s , O t t a w a . T h e a u t h o r s t h a n k

Dr . WITOLD JACHYMCZYK for h i s a s s i s t a n c e in t h i s work . T h e a u t h o r s a l so a c k n o w l e d g e

Merck , S h a r p a n d D o h m e for a s u p p l y of a c t i n o m y c i n D u s e d in t h e s e s t u d i e s . T h i s

r e p o r t is j o u r n a l p a p e r 3382 of t h e P u r d u e A g r i c u l t u r e E x p e r i m e n t S t a t i o n .

R E F E R E N C E S

i Y. MASUDA, Plant Physiol., 12 (1959) 325. 2 M. J. CHRISPEELS AND J. B. HANSON, Weeds, 4 (1962) 123. 3 J. L. KEY AND J. INGLE, Proc. Natl. Acad. Sci. U.S., 52 (1964) 1382. 4 J. SILBERGER AND F. SKOOO, Science, 118 (1953) 443. 5 R. ROYCHOUDHURY AND S. P. SEN, Plant Physiol., 17 (1964) 352. 6 S. C. I~¢[AHESHWARI, S. GUI-IA AND S. GUPTA, Biochim. Biophys. Acta, I17 (I966) 47 o. 7 J. H. CHERRY, Ann. N .Y . Acad. Sci., 144 (1967) 154. 8 G. R. CHANDRA AND J. E. VARNER, Biochim. Biophys. Acta, lO8 (1965) 583 . 9 M. M. JOHRI AND J. E. VARNER, Proc. Natl. Acad. Sci. U.S., 59 (1968) 269.

io D. J. OSBORNE, Plant Physiol., 37 (1962) 595. I I T. J. O'BRIEN, Ph.D. Thesis, Universi ty of Illinois, 1967. 12 R. C. C. HUAN6 AND J. BONNER, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1216. 13 W. J. JACHYMCZYK AND J. H. CHERRY, Biochim. Biophys. Acta, 157 (1968) 368. 14 R. J. MANS AND G. D. NOVELLI, Arch. Biochem. Biophys., 94 (1961) 48. 15 K. BURTON, Biochem. J., 62 (1956) 315 • 16 O. H. LOWRY, N. J. ROSEBROUGH, A. C. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951)

264 . 17 E. R. STOUT AND R. J. MANS, Biochim. Biophys. Acta, 134 (1967) 327 . 18 K. G. NAIR, M. RABINOWlTZ AND M. H. C. TU, Biochemistry, 6 (1967) i898. 19 J. H. CHERRY, H. CHROBOCZEK, W. J. G. CARPENTER AND A. RICHMOND, Plant Physiol., 4 °

(1965) 582. 20 J. D. MANDELL AND A. D. HERSHEY, Anal. Biochem., I (196o) 66. 21 K. A. O. ELLEM, J. Mol. Biol., 2o (1966) 283. 22 R. P. PERRY, Exptl. Cell Res., 29 (I963) 400. 23 S. LIAO, R. W. BARTON AND A. H. LIN, Proc. Natl. Acad. Sci. U.S., 55 (1966) 1593.

Biochim. Biophys. Acta, 169 (1968) 35-43