Effects of light on net Na+ and K+ transport in chlorella and evidence for in vivo cyclic phosphorylation

Plant Science Letters, 1 (1973) 405--41.1 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

EFFECTS OF LIGHT ON NET Na ÷ AND K ÷TRANSPORT C H L O R E L L A A N D E V I D E N C E F O R IN VIVO C Y C L I C PHOSPHORYLATION

IN

J. BARBER and Y.J. SHIEH*

Department of Botany, Imperial College, London, SW7 2BB (Great Britain) (Received May 4th, 1973)

SUMMARY

When K ÷ is added to a suspension of Chlorella pyrenoidosa grown so as to be abnormally rich in Na ÷ a net K÷/Na ÷ exchange occurs. In aerobic conditions the rate of exchange is only reduced to a b o u t 70% of the light rate while in an atmosphere of CO2-free N2 the light--dark difference is considerably increased mainly because of inhibition of the dark rate. The anaerobic photoinduced K+/Na ÷ exchange saturates with increasing light intensity and has an action spectrum corresponding to chlorophyll absorption. The net fluxes were slightly inhibited b y 3'-(3,4-dichlorophenyl)-l',l'~limethylurea (DCMU) b u t could be totally inhibited b y carbonyl cyanide m-chlorophenyl hydrazone (CCCP), 2,4-dinitrophenol (DNP) and N,N'-dicyclohexylcarbodiimide (DCCD). The results seem to indicate that under anaerobic conditions in vivo cyclic phosphorylation can support K+/Na ÷ exchange.

INTRODUCTION

The ability of b o t h photosynthetic and non-photosynthetic organisms to maintain internal levels of Na ÷ and K ÷ which are quite different to that o f their environment is well known. The maintenance of these ion gradients requires the utilisation of energy derived from metabolism. In the case of plant and algal cells the existence of active Na* extrusion and K ÷ accumulation has usually been concluded from experiments c o n d u c t e d under steady-state conditions 1,2. Recently, however, it was found that very large active net fluxes of Na ÷ and K ÷ could be induced with cells of ChloreUa pyrenoidosa 3,4. The * Present address: Institute of Botany, Academia Sinica, Nankang, Taipei (Taiwan). Abbreviations: CCCP, carbonyl cyanide m-chlorophenyl hydrazone; DCMU, 3'-(3,4-dichlorophenyl)-l', l'-dimethylurea; DCCD, N, hr-dicyclohexylcarbodiimide; DNP, 2,4dinitrophenol.

405

net fluxes were initiated b y adding K ÷ to a suspension of Chlorella cells grown so as to be rich in Na ÷ b u t depleted of K ÷. The object of this communication is to present and discuss experiments mainly designed to study the photosensitivity of the K+/Na ÷ exchange and to explore its possible use as a means of investigating in vivo cyclic phosphorylation. MATERIALS AND METHODS

Details of the procedures for culturing and harvesting Na+-rich ceils of Chlorella p y r e n o i d o s a have already been given 3. Net fluxes of K ÷ and Na ÷

were usually followed b y taking aliquots of cells and determining the cellular concentrations of these cations b y flame p h o t o m e t r y 3. However, for analyses of wavelength dependence of the photoinduced exchange, K 42 was used to measure the initial net K ÷ influx and again details of the procedure have already been published 4 The action spectrum for the K÷/Na ÷ exchange was measured using Balzer interference filters with half-band widths of 12.5 nm. Light was supplied b y a tungsten-halide lamp (Rank Aldis 2000 projector) coupled to a Zenith transformer. When measuring the effect o f various intensities of white light on the cation fluxes the distance between the light source and the reaction vessel was adjusted. Light intensities were measured with an ISCO spectroradiometer and were obtained by integrating the transmission spectra. Oxygen measurements were made with a Rank oxygen electrode (Rank Bros., Bottisham, Great Britain). All experiments were c o n d u c t e d at 25 + 0.5 ° either under aerobic conditions or in CO2 -free nitrogen. RESULTS T i m e course o f n e t K ÷ and N a ÷ m o v e m e n t

Fig. l ( a ) shows a typical time course of net K ÷ and Na ÷ m o v e m e n t when 3 mM KC1 is introduced into a K÷-free medium containing a suspension of Na÷-rich cells. It can be seen that these Na*-rich cells initially contained 74 mM Na ÷ and 20 mM K ÷ b u t after 90 min the Na ÷ level had decreased to 5~mM while the internal K ÷ concentration had risen to a b o u t 100 raM. K ÷ uptake and Na ÷ release b o t h appear to be controlled b y a single exponential function of time and the curves through the experimental points in Fig. l ( a ) have been drawn according to the first-order equations shown. The time constants of 10.1 rain for N a ÷ efflux and 11.4 rain for K ÷ influx and the initial concentration terms of 65.4 m M N a ÷ and 82 m M K + were c o m p u t e d from the least square analyses of the data plotted semi-logarithmically as s h o w n in Fig. l(b). The initialrates for this experiment correspond to a net K ÷ uptake of 7.2 mequiv. K+/min. I cell water and a net N a ÷ efflux of 6.5 mequiv. Na÷/min •l

406

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Fig. 1 (a) N e t N a + e x t r u s i o n ( v ) a n d K + u p t a k e (o). T h e fluxes w e r e m e a s u r e d w i t h illumin-ated cells b u b b l e d w i t h m o i s t air a n d k e p t a t 25 ° ±: 0.5 ° . T h e curves were d r a w n - a c c o r d i n g t o t h e equatioris s h o w n w h i c h were derived using least squares analyses o f t h e d a t a p l o t t e d s e m i - l o g a r i t h m i c a l l y as s h o w n in (b).

cell water. The imbalance in charge movement was always f o u n d to occur and could be accounted for by K* induced net H ÷ efflux 4

Effect o f light on IC/Na +exchange Under aerobic conditions the net K + and Na t fluxes were photosensitive but the dark rate was usually about 70% of the light rate (Table I). However, as Table I shows when the net fluxes were measured in an atmosphere of CO2free nitrogen the difference between the light and dark values was enhanced. The reason for this was that under this gaseous condition the da~k rates of TABLE I E F F E C T O F C O 2 - F R E E N2 O N N E T F L U X E S O F K ÷ A N D Na t M E A S U R E D IN D A R K OR LIGHT CONDITIONS Net K ÷ influx

Air CO 2 -free N 2

N e t Na t e f f l u x

Light

Dark Light ( m e q u i v . / m i n : l cell w a t e r )

Dark

7.92 6.00

5.28 1.28

4.88 0.88

7.00 4.88

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net exchange were considerably reduced suggesting a dependence on respiration. In contrast, CO2 -free nitrogen had less effect on the light rates indicating that the net cation exchange can be powered b y a process independent of CO2 fixation and possibly more closely associated with primary electron transport in photosynthesis. The intensity of the illumination affected the extent of the light-induced exchange under anaerobic conditions (Fig. 2) and it was found that both net fluxes saturated at the same light intensity. In order to check if the photoinduced fluxes were mediated b y photosynthesis an action spectrum was measured. As Fig. 3 shows, when net K ÷ influx was measured at a fixed nonsaturating light intensity (1 mW. cm-2 ) at different wavelengths an action spectrum was obtained which strongly implicates chlorophyll as the main p h o t o a c c e p t o r in promoting the K÷/Na ÷ exchange. Estimates of relative quantal efficiencies indicated that far-red light absorbed by the cells was slightly more effective than short wavelength light.

Effect of DCMU As fig. 4 shows under aerobic conditions concentrations of DCMU which totally blocked 02 were found t o reduce the K÷/Na ÷ exchange to a b o u t 80%.

Effect of CCCP, DNP and DCCD The Na÷/K ÷ exchange was sensitive to the uncouplers CCCP and DNP and to the energy transfer inhibitor DCCD. Concentrations for 50% and 100% inhibition are shown in Table II.

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Fig. 2. E f f e c t o £ different white light intensities produced by an incandescent light source working at full voltage o n t h e i n i t i a l rates o f net Na + extrusion (v) and n e t K ÷ u p t a k e (o). The cells were bubbled with CO 2 -free N 2 and the dark rate has been substraeted from the rates obtained in the light.

408

4

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I 650 Wavelength (nm)

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Fig. 3. Effects o f different wavelengths of light on the light-dependent initial rate of K ÷ uptake. Light intensity at all wavelengths was 1 m W . c m -2 which was below saturation. O t h e r c o n d i t i o n s as given for Fig. 2.

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Fig. 4. E f f e c t o f D C M U on o x y g e n e v o l u t i o n (A), net Na ÷ e x t r u s i o n (e) and n e t K ÷ u p t a k e ( , ) . T h e net cation e x c h a n g e was measured using illuminated cells bubbled w i t h CO 2 -free N a while o x y g e n e v o l u t i o n was measured under aerobic conditions.

409

T A B L E II INHIBITION OF K+/Na ÷ E X C H A N G E BY CCCP, DNP AND DCCD Measured with illuminated cells u n d e r aerobic c o n d i t i o n s Compound

CCCP DNP DCCD

Inhibition 50% (M)

100% (M)

10-s 10 -4 10 -4

5 . 1 0 -s 5 . 1 0 -4 5 . 1 0 -4

DISCUSSION

The effect of inhibitors of phosphorylation indicates that the K÷/Na ÷ exchange is an active process powered by ATP. The low sensitivity of the K÷/Na~ exchange to illumination under aerobic conditions probably reflects the high rate of respiration relative to the rate of photosynthesis found with Na÷-rich cells 3. This would mean that in illuminated aerobic conditions the rate of ion transport is determined by the activity of the pump rather by the limitations imposed by the ATP synthesising system. However, under anaerobic conditions the dark rate is suppressed presumably because of the inhibition of oxidative phosphorylation. The stimulatory action of light when cells were bubbled with CO2 -free N2 would implicate cyclic photophosphorylation or possibly oxygen-catalysed non-cyclic photophosphorylation (often called pseudocyclic phosphorylation). Certainly there is good evidence that the photo-induced exchange is linked to partial reactions of photosynthesis. The exchange showed light saturation and had a "chlorophyll-like" action spectrum. Similar observations have been reported for active cation transport in Hydrodictyon africanum s. The slight reduction of the light-induced exchange under anaerobic conditions by DCMU could mean that only a small portion of the energy source was derived from pseudocyclic election flow. Alternatively there is always the possibility that inhibition of photosystem two by DCMU induced a switch from oxygen-catalysed pseudocyclic flow to cyclic election flow. Nevertheless, there seems little doubt from the high quantal efficiency of far-red light and lack of significant inhibition of anaerobic exchange by DCMU that cyclic phosphorylation can occur in vivo under some conditions. The question whether such a process is important when photosystem two is also operating (that is at short wavelength light and in the absence of DCMU) is still unanswered. From studies with intact cells of Hydrodictyon, Raven s has argued that both processes can occur when the two photosystems are operating. He has suggested from studies of active K ÷ movement that there is a control mechanism in intact cells which under some conditions prevents pseudocyclic election flow and accounts for the high relative quantal efficiencies of ATP-requiring processes measured in the far-red (beyond 680 nm). 410

Overall the rapid first-order exchange o f K ÷ for Na ÷, detected When K ÷ is added to a suspension of Na÷-rich Chlorella cells, is an energy-requiring process which seems to be an ideal p h e n o m e n o n for a much more detailed investigation of the existence and properties of in vivo cyclic and pseudocyclic photophosphorylation. In this respect experiments which affect the redox states of electron acceptors in photosystem one, for example, involving CO2 -enriched N : , may be enlightening (see ref. 6). ACKNOWLEDGEMENTS

The work was supported by the Science Research Council and the Research Fund of the University of London and carried out while one of us (Y.J.S.) held an International Atomic Energy Agency Fellowship. REFERENCES 1 J. Dainty, Ann. Rev. Plant Physiol., 13 (1962) 379. 2 E.A.C. MacRobbie, Quart. Rev. Biophys., 3 (1970) 251. 3 Y.J. Shieh and J. Barber, Biochirn. Biophys. Acta, 233 (1971) 594. 4 J. Barber and Y.J. Shieh, J. Exptl. Botany, 23 (1972) 627. 5 J.A. Raven, New Phytol., 68 (1969) 45. 6 J.A. Raven, J. Exptl. Botany, 22 (1971) 420.

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