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Effect of C 0 2 on Photophosphorylation in vivo as Revealed by the Light-dependent Cle Uptake in Elodea densa

W . D . J E S C H K E a n d W . S I M O N I S

Botanisches Institut der Universität Würzburg

( Z . Natur f o r s chg . 22 b, 8 7 3 — 8 7 6 [ 1 9 6 7 ] ; e i n g e g a n g e n am 4. F e b r u a r 1967)

The light-dependent Cl e uptake in Elodea densa exhibits dual DCMU inhibition curves depen-dent on the presence and absence of C02 . This suggests that cyclic photophosphorylation (energy surce in the absence of C02) is also inhibited by higher concentrations of DCMU in vivo. Further, at low DCMU concentrations the Cl0 uptake in the presence of C02 is much smaller than would be expected if cyclic photophosphorylation were operative. This points to a parallel inhibition of non-cyclic and cyclic photophosphorylations by low concentrations of DCMU in the presence of CO,. The apparent inhibition of cyclic photophosphorylation at low concentrations of DCMU can be reversed by withdrawal of C0 2 . It is concluded that C02 may be important in the regulation of cyclic and non-cyclic photophosphorylations in vivo.

The occurrence in vivo of cyclic photophosphory-lation besides the non-cyclic type is documented in several organisms 1 - 5 mostly by the existence of a photophosphorylation that is not inhibited by DCMU *. However, little is known about the relative amounts of both types of photophosphorylation inside the cell and their mutual regulation. In a pre-vious paper the light-dependent C l 0 uptake in Elo-dea densa during 30 — 40 min was shown to be sup-ported by photophosphorylation6 . C l e uptake in Elodea seems to be a useful test reaction for non-cyclic and cyclic photophosphorylation in vivo7'6

since light enhances the C l 0 uptake manifold as compared to darkness under conditions in which non-cyclic and cyclic or only cyclic photophosphory-lations are possible.

The present report gives evidence that the light-dependent Cl 0 uptake in air exhibits different DCMU inhibition curves dependent on the presence and absence of C 0 2 . Further it is suggested that C 0 2 may play an important part in the regulation of cyclic and non-cyclic photophosphorylations in vivo as proposed by A R N O N 8 ' 9 . The results pre-sented here lend further support to the v iew 6 that ATP synthesis rather than photosynthetic electron transport10 sustains the active C1G transport in the light in Elodea contrary to the events in Nitella.

1 G. FORTI and B. P A R I S I , Biochim. biophysica Acta [Amster-dam] 71,1 [1963].

2 W. SIMONIS, Ber. dtsch. bot. Ges. 77, (5) [1964]. 3 W. URBACH and W. SIMONIS, Biochem. biophysic. Res. Com-

mun. 17,39 [1964]. 4 W . T A N N E R , L . DÄCHSEL, and 0. K A N D L E R , Plant Physiol.

40,1151 [1965]. 5 W. NULTSCH, Photochem. Photobiol. 4, 613 [1965]. * DCMU = 3- (3,4-dichlorophenyl) 1,1-dimethylurea.

Material and methods

Elodea densa was grown in an artificial growth me-dium 11 and illuminated in a 14/10 hours light and dark cycle. The growth medium contained 10~5 M C l 0

ions. ClQ uptake. Leaves of Elodea were detached and

kept overnight in the dark in an inactive buffered solu-tion corresponding to the reaction mixture but con-taining 1 0 _ 5 M Cl 0 ions. In the same (renewed) solu-tion the leaves were pretreated for 40 min under the experimental conditions (temperature, light/dark, aera-tion). The uptake of 36C10 was started by exposure of the leaves to 10 ml reaction mixture containing in moles//: C l 0 : 2 x 10~4, Na®: 7,56 x l 0 ~ 4 ; K®: 1,84 x l 0 ~ 4 ; Ca2®: 1 x 10~4 ; M g 2 ® : l x l 0 - 5 ; SO 4 2 0 : 1,4 x l 0 ~ 4 ; H 2 P0 4 ®: 1 x 10~5 ; TRIS: 3 , 3 x 1 0 - " ; phthalate:

6,7 x 1 0 - 4 ; and 36C1® at a specific activity of 22 ^Ci/mmole C l 0 . The ph was 5.2. The temperature was maintained at 25 °C.

The solution was aerated and thus stirred with the gas mixtures as specified in the legends of figs. 1 and 2. The uptake of 3 6Cl e was terminated by thorough washing of the leaves with 5 x 10~4 M NaCI solution. The leaves were fixed to adhesive tape on planchets, dried, and counted with a thin window methane flow counter. The leaf area was determined by weighing. DCMU was added in methanol solution yielding a final concentration of 1% methanol that was also present in the controls. The photosynthetic 02-production was measured polarographically. For details see 1. c. 6.

6 W. D . JESCHKE, Planta 7 3 , 1 6 1 [ 1 9 6 7 ] . 7 W. SIMONIS, Ber. dtsch. bot. Ges., im Druck [ 1 9 6 7 ] . 8 D . I . A R N O N , i n : J . B . THOMAS a n d J . C . GOEDHEER, E d s . ,

Currents in Photosynthesis, Rotterdam 1966. 9 K . T A G A W A , H. Y. TSUJIMOTO, and D. I. A R N O N , Nature

[London] 1 9 9 , 1 2 4 7 [ 1 9 6 3 ] . 10 E. A. C. M A C R O B B I E , Biochim. biophysica Acta [Amster-

d a m ] 9 4 , 6 4 [ 1 9 6 5 ] . 11 W. D . JESCHKE U. W. SIMONIS, Planta 6 7 , 6 [ 1 9 6 5 ] .

Results and discussion

1. DCMU action on non-cyclic photophosphory-lation. Fig. 1 presents the concentration curves of DCMU action on C l e uptake in Elodea leaves in the light and in darkness. Curve A, measured in air + 3% C 0 2 , strongly resembles the inhibition curve

of 02-production in Elodea6 as follows also from the similar concentrations of half inhibition

[DCMU] [mol/l]

Fig. 1. Inhibition by DCMU of Cl° uptake in leaves of Elo-dea densa as a function of DCMU concentration. A. light, 4000 Lux, air+ 3% C02 , B. light, 4000 Lux, air without C0 2 , C. darkness air (0,03% C02). Each point corresponds to the

average of 8 or 16 (curve B) determinations.

(table 1 ) . It can be concluded that in the presence of abundant C 0 2 the active C l e uptake is supported energetically to a great extent by non-cyclic photo-phosphorylation. This finding seems important inasmuch as it implies that ATP produced by non-cyclic photophosphorylation is contributed to a pool of ATP that is also available for processes different from COo-assimilation.

The uninhibited ca. 30% of CI® uptake (curve A ) may be sustained either by cyclic photophosphory-lation or by oxidative phosphorylation, since the depression of oxidative phosphorylation in the light 2 does not necessarily remain, if photosynthesis is inhibited by DCMU.

2. DCMU inhibition of oxidative phosphorylation and cyclic photophosphorylation. As is seen from fig. 1, DCMU at high concentrations also affects the C l 0 uptake in the dark (curve C ) , probably by an unspecific action on oxidative phosphorylation that may be compared to the effects of DCMU in Anki-strodesmus 7 and in Phormidium 5.

1 2 M . LOSADA a n d D . I . A R N O N , i n : R . M . HÖCHSTER a n d J . H . QUASTEL, Eds., Metabolic Inhibitors, Academic Press, New York 1963.

13 A. V. T R E B S T and H . E C K , Z. Naturforschg. 16 b, 4 5 5 [ 1 9 6 1 ] .

A B Reactions and conditions Ci50 Cl-uptake

[DCMU] 10-9 mole [mole/1] em2 • h

0 2 -production light, 4000 Lux 1,7 X lO-7

Cl- uptake, air, 3% CO2, light, 4000 Lux 2,5 X lO-7 6,4 (23)

Cl- uptake, air, no CO2, light, 4000 Lux 1,1 x 10-6 7,5 (24)

Cl- uptake, N2 , no CO2, light, 4000 Lux 1,5 X 10-6

Cl- uptake, air (0,03% C02) darkness 1,4 X lO"4 L6 (8)

Table 1. A. DCMU concentrations (Ci 50) effecting 50% in-hibition in several reactions of Elodea leaves; the figures were obtained as points of inflection of the inhibition curves. B. Ab-solute values of Cl0 uptake (influx) of Elodea leaves in 2 x 10 - 4 M Cl® solution. Averages of (n) determinations.

However, in the light a second inhibition curve of DCMU action can be obtained if the C l e uptake is measured in the absence of C02 in air (fig. 1, curve B) or in N 2 6 . Under these condition only cyclic (N2 ) or cyclic and oxidative phosphorylations (air without C 0 2 ) are possible. Since oxidative phosphorylation is not influenced except at much higher concentrations (fig. 1) it seems evident that in vivo also cyclic photophosphorylation is inhibited by DCMU as was also found in Chlorella4. The relative sensitivities of C l e uptake in the presence (non-cyclic photophosphorylation) and in the ab-sence of C 0 2 (cyclic photophosphorylation) differ only by a factor 10 in DCMU concentration (fig. 1, table 1 ) .

In isolated chloroplasts DCMU is without effect on cyclic photophosphorylation according to A R N O N 12 and to T R E B S T 13. On the other hand, A S A H I

and J A G E N D O R F 14 suggest a second site of DCMU action active in cyclic photophosphorylation. It is possible, however, that cyclic photophosphorylations as measured in vitro with the unphysiological phen-azine methosulfate (PMS) or with large concentra-tions of ferredoxin 15 as cofactors do not give a true picture of the events in vivo 16. Thus the affinity of DCMU to the PMS-mediated electron path may be smaller than to the cyclic electron flow in vivo. Also

1 4 T . A S A H I and A . T . JAGENDORF, Arch. Biochem. Biophysics 100,531 [1963].

15 D. I . A R N O N , Science [Washington] 1 4 9 , 1460 [1965]. 1 6 L . N. M. DUYSENS, Progr. Biophysics biophysic. Chem. 1 4 ,

1 [1964].

it is possible on the grounds of mass action that the high concentrations of ferredoxin as used in vitro require a greatly increased DCMU concentration for an inhibition.

3. Suppression of cyclic photophosphorylation at low DCMU concentrations in the presence of C0.2 . The absolute values of Cl® uptake in the presence and in the absence of C 0 2 are almost the same1 7

(table 1 ) . Thus the inhibited values in fig. 1 can be directly compared and it follows that at 5 x 1 0 - 7 M DCMU the Cl® uptake in the absence of C 0 2 (cyclic photophosphorylation) is considerably higher than in the presence of C 0 2 (fig. 1 ) . Under the provision that Cl® uptake is supported by photophosphoryla-tion this implies that uninhibited cyclic photophos-phorylation could sustain a substantially higher Cl® uptake than is actually found after inhibition by DCMU in the presence of abundant C 0 2 . Apparent-ly at the low concentration (5 X 1 0 - 7 M ) of DCMU not only non-cyclic but also cyclic photophosphory-lation is greatly decreased in the presence of abun-dant C 0 2 . At least there is no compensation by cyclic photophosphorylation for the non-cyclic type after inhibition of the latter by DCMU — as was also postulated for Chlorella by G O U L D and B A S S H A M 18 for other reasons.

Apparently quite different situations obtain in vivo if the non-cyclic electron transport is suppres-sed at the reductive end of the electron chain (by withdrawal of C 0 2 ) or at the oxidative end (by DCMU) provided that C 0 2 is present. In the first case cyclic photophosphorylation still is possible and probably enhanced, in the second case it seems greatly to be suppressed too.

According to T A G A W A et al. and to A R N O N 8 ' 9 the regulation in the cell between non-cyclic and cyclic photophosphorylation could be effected by the re-duction state of NADP. On the basis of this scheme the results of fig. 1 can be explained: In case the noncyclic electron transport is inhibited by lack of C 0 2 , ferredoxin and NADP are not oxidized by C 0 2 . In the light they are continuously maintained in a reduced state. Thus an excess of electrons can flow bade to some intermediate of the electron chain and thus permit a cyclic electron transport. If on the

other hand the non-cyclic electron transport is in-hibited at the site of oxygen evolution by DCMU in the presence of C 0 2 , then the few electrons ar-riving at ferredoxin or NADP will be consumed by the reduction of C 0 2 leaving NADP in an oxidized state and an electron gap the site of ferredoxin or the substance Z 19 of light reaction I. Thus cyclic electron transport cannot proceed to a great extent although it is not specifically inhibited by DCMU at this low concentration.

Starting from this explanation it can be predicted that the apparent inhibition of cyclic photophospho-rylation at 5 x l O ~ 7 M DCMU in the presence of C 0 2 should be abolished if only after the inhibition C 0 2 is removed.

300

200

100

2 leaf

i

-C02 ? 1* DCMU)/

i +-+3% CO

A / i

/ i KO/ \i

/ //+3%C02 // (+DCMU)

/ / 1

//5-10~7m I AS DCMU ! r i i i : i i i i

20 40 60 SO t [min]

Fig. 2. Reversal of DCMU inhibition of light-dependent C l e

uptake in Elodea densa leaves by withdrawal of C02 . 36C1Q

uptake was measured first in the presence of 3% C02 in air in the light (4000 Lux) at a concentration of 5 x 10~7 M DCMU (control "Ko" without DCMU). In a part of the samples the gassing was changed afterwards to air without C0 2 , the others still getting air+C0 2 . C1Q uptake was measured for a second 40 min period in the presence of the same DCMU

concentration.

The results of an appropriate experiment are shown in fig. 2. Elodea leaves were first treated with DCMU in the presence of 3% C 0 2 resulting in an inhibited Cl ® uptake. After 40 min in a part of the experimental vessels the gassing was changed to air without C 0 2 . As is seen from fig. 2 the withdrawal of C 0 2 completely reverses the DCMU inhibition: The leaves without C 0 2 exhibit an uptake of Cl®

The somewhat lower value in the presence of C02 points to a competition between C02 assimilation and Cl0 uptake probably for ATP 6. At low light intensities C02 consider-ably inhibits the Cl0 uptake in the light 6.

1 8 E . S . G O U L D and J . A. BASSHAM, Biochim. biophysica Acta [Amsterdam] 102, 9 [ 1 9 6 5 ] .

1 9 H . T . W I T T , B . R U M B E R G , P . S C H M I D T - M E N D E , U . SIGGEL, B . S K E R R A , J . V A T E R , and J . W E I K A R D , Angew. Chem. 7 7 , 8 2 1 [ 1 9 6 5 ] .

more than twice as large as in the parallels with COo although both samples still are incubated with 5 x 10~ 7 M DCMU.

We interpret this result as proof of a resumption of cyclic photophosphorylation which now is pos-sible again as electrons arriving at ferredoxin are no

longer drained away by reduction of NADP or C 0 2 .

This investigation was supported by the D e u t -s c h e F o r s c h u n g s g e m e i n s c h a f t . We greatly appreciate the skilful technical assistance of Miss GABRIELE W O L F F .

Enzyme der Polynucleotid-Synthese in pflanzlichen Organismen

II. Isolierung, Reinigung und Charakterisierung der RNA-Polymerase aus der Blaualge Anacystis nidulans

I N G R I D C A P E S I U S u n d G E R H A R D R I C H T E R

Botanisches Institut der Universität Tübingen

(Z. Naturforschg. 22 b, 876—885 [1967] ; eingegangen am 10. Februar 1967)

The isolation and purification of RNA polymerase from the blue-green alga Anacystis nidulans is described. The method consists in an initial fractional centrifugation which yields 60 — 70% of the enzyme associated with a DNA-rich fraction. It is subjected to column chromatography (DEAE-cel-lulose) and gel filtration (Sephadex G-200) to obtain a 110 —120 fold increase in specific activity over the crude extract. The preparation is free from polynucleotide phosphorylase, kinases, phos-phatases and nucleic acids. The enzyme requires DNA, a primer, as well as Mg2®, Mn2® and the 4 ribonucleoside triphosphates for the net synthesis of polyribonucleotides. Salmon sperm DNA and native DNA from Chlorella pyrenoidosa can replace Anacystis DNA as primer. The properties of the product, however, depend to a certain extent on the primer DNA used as revealed by column chromatography of the former on methylated serum albumin. After isolation by this method, the synthesized polyribonucleotides were found to be partially stable against the action of RNase. This finding suggests a possible association of the newly synthesized RNA with the primer DNA in form of a stable complex which may function as an intermediate of the DNA directed RNA synthesis.

Ein wichtiges Enzym für die Biosynthese von Ribopolynucleotiden ist die RNA-Polymerase ( „Nu-cleosidtriphosphat : RNA-Nucleotidyltransferase", E. C. 2 .7 .7 .6 ) . Es benötigt als Substrat die vier Nu-cleosidtriphosphate ATP, GTP, CTP und UTP sowie hochmolekulare DNA als „Starter". Die letztere ist für die Anordnung der Nucleoside in der entstehen-den Ribopolynucleotid-Kette verantwortlich, da sie als Matrize wirkt. Nachweis und Reinigung der RNA-Polymerase ist bei einer Reihe von Bakterien und bei einigen tierischen Objekten gelungen. Uber ihr Vorkommen und ihre Eigenschaften bei Pflanzen sind wir hingegen nur spärlich informiert. Die bis-herigen Untersuchungen beschränken sich auf nie-dere Photosynthese-Organismen1 und Keimlinge höherer Pflanzen2. Wir haben daher den Versuch unternommen, RNA-Polymerase aus einer Blaualge, Anacystis nidulans, anzureichern und ihre Eigen-

1 G. RICHTER. Biochim. biophysica Acta [Amsterdam] 7 2 , 3 4 2 [ 1 9 6 3 ] .

2 J. R. MANS and G. D. NOVELLI, Biochim. biophysica Acta [Amsterdam] 91, 1 8 6 [ 1 9 6 4 ] .

schaften zu untersuchen. Einige vergleichende Ex-perimente wurden an der einzelligen Grünalge Chlo-rella pyrenoidosa durchgeführt.

Wie bereits in einer früheren Publikation 3 berich-tet wurde, basiert das gewählte Verfahren auf der Beobachtung, daß die RNA-Polymerase offensichtlich mit der DNA der Zelle assoziiert ist und durch eine fraktionierende Zentrifugation des Zellhomogenats mit dieser als subcelluläre Fraktion angereichert wer-den kann. Diese stellt das Ausgangsmaterial für die weitere Reinigung des Enzyms dar. Das andere in Anacystis möglicherweise aktive Enzym der Ribo-polynucleotid-Synthese, nämlich die Polynucleotid-Phosphorylase, wird bei diesen einleitenden Zentri-fugier-Schritten größtenteils mit den Ribosomen in einer anderen Sedimentfraktion abgetrennt.

Neben den Bedingungen, die zur vollen Aktivität der RNA-Polymerase erfüllt sein müssen, galt unser

3 I . CAPESIUS U. G . RICHTER, Z . Naturforschg. 2 2 b. 2 0 4 [ 1 9 6 7 ] .