Photosynthesis Research 11:109-118 (1987) © Martinus Nijhoff Publishers, Dordrecht - - Printed in the Netherlands

109

Regular paper

Observation and characterisation of a transient in the yield of chlorophyll fluorescence in intact spinach chloroplasts K.A. CARVER 1 and P. HORTON 1'2 Research Institute for Photosynthesis1 and Department of Biochemistry~, The University, Sheffield, S10 2TN, UK (Received: 3 February 1986; in revised form: 24 March 1986 )

Key words: photosynthesis, chloroplast, chlorophyll fluorescence Abstract. A transient in chlorophyll fluorescence, which is associated with a transient in 9-aminoacridine fluorescence and a perturbation in the rate of oxygen evolution, has been observed in intact spinach chloroplasts. The results indicate that changes in the redox state of Q are, at least partially, responsible for the transient in chlorophyll fluorescence. The size of the transient is highly dependent upon the concentration of inorganic phosphate and upon the pH of the medium. The properties of the transient are consistent with the suggestion that it reflects changes in the levels of stromal intermediates during induction. Abbreviations.

BES: NN-Bis(2-hydroxyethyl)2-aminoethanesulphonicacid dihydroxyacetone-P(DHAP): dihydroxyacetone phosphate glycerate-3-P (PGA): glycerate-3-phosphate HEPES: N-2- Hydroxyethylpiperazine-N-2-ethanesulphonicacid MES: 2-(N-Morpholino)ethanesulphonic acid Pi: inorganic phosphate qE: quenching of chlorophyll fluorescence by the energisation of the thylakoid membrane qQ: quenching of chlorophyll fluorescence by oxidised Q, the electron acceptor of photosystem 2 ribose-5-P (R5P): ribose-5-phosphate Rbu-5-P: ribulose-5-phosphate Introduction

A wide range o f studies using chloroplasts [2, 3, 7 ] , protoplasts [10, 11] and leaves [14, 15, 19, 21, 22] have established a clear relationship between photosynthetic carbon assimilation and the quenching of chlorophyll fluorescence. This relationship exists primarily because the yield of fluorescence is determined by the redox state of the reaction centre o f photosystem 2 (termed qQ quenching) and the size o f the transthylakoid ApH (qE). These, in turn, are influenced by the respective turnover rates o f NADPH and ATP by carbon assimilation [for reviews see 5, 15]. Upon illumination o f these systems an induction period is observed before photosynthesis reaches steady state. The kinetics of photosynthesis during the induction period depend on factors such as light intensity and temperature as well as on the light

110 treatment preceding illumination. In leaves and protoplasts following a long dark period a single oscillatory transient, both in the quenching of fluorescence and the acceleration of 02 evolution, was observed [10]. The oscillations can be multiple and of steadily decreasing amplitude upon reillumination after short dark intervals [21, 22]. Analysis of the fluorescence signal [11] or the use of light scattering as a probe of ApH [16] demonstrate that changes in both qQ and qE accompany the oscillations in photosynthesis. It has been suggested that the oscillatory behaviour results from the operation of regulatory mechanisms in carbon assimilation [15, 18, 19]. In intact chloroplasts, however, much simpler induction kinetics have been described. The rate of 02 evolution accelerates exponentially after a lag, this rise being entirely non-oscillatory. Similarly, chlorophyll fluorescence is quenched continuously during induction [3, 7]. The acceleration in O2 evolution, causing increased qQ quenching, is also associated with a fall in ApH which leads to a relaxation of qE quenching. Sometimes these events can cause a shoulder on the fluorescence quenching curve as qQ and qE are changing in opposite directions [2, 5]. This antiparallel relationship between qQ and qE occurs because of relief of a limitation on ATP consumption and is most graphically seen upon addition of glycerate-3-P to the reconstituted chloroplast system [3, 6]. It is different to that seen during oscillation when qQ and qE apparently act in concert, probably because of limitation by ATP production. This latter behaviour has also been simulated in the reconstituted chloroplast system by addition of ribose-5-P [3, 6]. The apparent absence of oscillatory behaviour in intact chloroplasts suggests that cytoplasmic events exert a strong influence upon induction kinetics. More specifically, export of triose phosphate in exchange for Pi in intact chloroplasts via the phosphate translocator may well prevent fluctuations in the levels of reductive pentose phosphate pathway intermediates and in the adenylate pool, hence dampening oscillatory behaviour. Recent work with leaves has in fact shown that a decrease in cytoplasmic phosphate level imposed by mannose feeding increases the tendency (i.e. lowers the light intensity and or COs concentration required) for oscillatory behaviour [20]. In this paper it is demonstrated that exclusion of Pi from the reaction medium results in the appearance of oscillatory behaviour in intact chloroplasts. Materials and methods

Intact chloroplasts were isolated as described previously [17] with the exception that 57 mM MES was used to buffer the isolation medium. Unless otherwise stated, intact chloroplasts (47-75/ag Chl.m1-1) were assayed with sorbitol (330mM), EDTA (2mM), MgCI2 (lmM), MnC12 (lmM), Tricine (50mM), NaHCOa (10mM), Catalase (220 U/ml) and 9-aminoacridine

111

A:100pM R

]', r.. I I~ ,

p, I! I!

I

t

C: No Pi

B: 50pM Pi

P,

lmin

I

I

I!l I

x I

Ii I: I



X

I I

x

....

.c

• "•...- . . . . . . . . . . . .

":"............................

8

•.." ........

t

I I jill

~~

t

o J i i a ~



~

......

o 3

t

On

t

On

t

On

Figure. 1. Measurement of oxygen evolution ( - - ) , chlorophyll fluorescence (----) and 9-aminoacridine fluorescence ( . . . . ) at different levels of inorganic phosphate. The maximum rates of 02 evolution were 104, 93 and 77 #moles O2/mg chl/h in A, B and C respectively• The assay medium included tricine buffer at pH 8.1. 'On' refers to switch on of both actinic light and fluorescence measuring beam. (5/aM) adjusted to pH 8.1. Oxygen evolution, chlorophyll fluorescence and 9-aminoacridine fluorescence was measured simultaneously in a modified Hansatech 02 electrode as described b y Horton [3].

Results Intact chloroplasts, evolving 02 in the presence o f saturating bicarbonate at pH 8.1, exhibited a secondary transient increase in chlorophyll fluorescence (Figure 1). This transient was most obvious when the chloroplasts were

112

~""... "% "....

pH7.8 ""'"

%'°......

' ~ 6"'..%.. 9

..%%

""-o.. : '"..

t

~ p H 7 - 2

~

'",....

~

"'".....,. ~- ",... ".. ®~ ~-o 8 == 0

"%.. ".,oo.%.

.

i".. ".. pH7'5

PH8"4

%°.

%"-.°..,

u=

pH 8'1

%°.,,°

\:XJ

0.05

°'%° %%..o%

I

pH8-7

lmin I

1'

t

On

On

Figure 2. Effect of pH upon the yield of chlorophyll fluorescence ( . . . . ) and oxygen evolution ( ). Conditions as in Figure 1C. The pH was varied by using 50 mM BES (pH 6.9 and 7.2) or 50 mM HEPES (pH 7.5 and 7.8) in place of trieine.

assayed in the absence of added Pi, where it can be seen to be associated with changes in the ApH (measured by 9-aminoacridine fluorescence) and in the change in the rate of oxygen evolution. The secondary rise in 9-aminoacridine fluorescence clearly precedes that in chlorophyll fluorescence. It shoud be noted that the transient is superimposed upon a gradual decline in photosynthesis as the pool of Pi is depleted.

113

...

E

-

/

.'/ ....

...

i /

.

.: ~

m

DCMU

J= (.3

I

2 min

I

Figure 3. Measurement of the contribution of qE and qQ to the chlorophyll fluorescence induction curve. 10 uM DCMU was added where indicated (t) to different chloroplast samples; the rapid fluorescence rise reflects the extent of qQ quenching and the slower rise that of qE. Conditions as in Figure 1C. The transient in chlorophyll fluorescence is highly dependent upon the pH of the medium (Figure 2). In the absence of added Pi, the transient only becomes obvious when the pH of the medium is above 7.8. At pH values below 7.8 fluorescence quenching is slower and the induction period is longer. The relative contributions of qQ and qE to chlorophyll fluorescence quenching can be assessed from the changes in chlorophyll fluorescence which occur upon addition of DCMU [7]. This technique was employed to characterise the quenching components involved in the chlorophyll fluorescence transient (Figure 3). DCMU blocks electron transfer from Q to plastoquinone and consequently the rapid rise in chlorophyll fluorescence, occuring upon addition of DCMU, shows the further reduction of oxidised Q and represents the extent of qQ quenching. The slower rise in chlorophyll fluorescence reflects the dissipation of the proton gradient following the inhibition of

114 electron transport and is therefore indicative of the extent of qE quenching. It can be seen that, although representing only a small proportion of the total quenching, relatively large changes in qQ are involved in the chlorophyll fluorescence transient. The transient is reflected in the small differences in the heights of the peaks of the fast DCMU-induced rise indicating a minor contribution from changes in qE quenching; the extent of change in qE quenching is smaller despite its large contribution to the overall fluorescence quenching. As found in previous studies [3, 7, 11] there is an additional component of fluorescence quenching which is not due to qQ or qE and which appears not to exhibit oscillitory behaviour. The effects of adding reductive pentose phosphate pathway metabolites upon chlorophyll fluorescence is shown in Figure 4. The addition of 2 mM glycerate-3-P increased the rate of chlorophyll fluorescence quenching as previously shown [3] ; the transient was decreased in amplitude and brought forward. The transient in 9-aminoacridine fluorescence and the rate of 02 evolution were suppressed. Dihydroxyacetone-P and ribose-5-P both affected the chlorophyll fluorescence signal in the same way; the initial rate of quenching, together with the amplitude of the transient, being decreased although the timing of the transient was not obviously affected. Both dihydroxyacetone-P and ribose-5-P decreased the extent of 9-aminoacridine fluorescence quenching as well as displacing this transient to an earlier position. In contrast, dihydroxyacetone-P and ribose-5-P had different effects upon oxygen evolution. Dihydroxyacetone-P increased the length of the induction period but otherwise had little effect upon the kinetics of oxygen evolution. Ribose-5-P, on the other hand, had little effect upon the induction period but decreased the initial rapid rate of oxygen evolution to such an extent that any subsequent perturbation in rate was barely detectable. Discussion The transient in chlorophyll fluorescence reported here in intact chloroplasts resembles, by virtue of being associated with a perturbation in the rate of oxygen evolution, those transients which have been described for leaves and protoplasts [10, 21, 22]. Measurements of light scattering [16] and qE [11] quenching had suggested an involvement of ApH changes. However these are only indirect methods for the estimation of ApH and it is significant therefore that a more direct method, namely the measurements of 9-aminoacridine fluorescence, shows the occurrence of changes in ApH. Furthermore, 9.aminoacridine fluorescence responds rapidly to ApH change whereas chlorophyll fluorescence changes have a slower time constant [e.g. see 3]. Use of intact chloroplasts therefore allows a more reliable estimate of the timing of transient events to be made. The data presented here clearly show that the first observable event in the transient is the rise in 9-aminoacridine fluorescence; the rise in cMorophyU fluorescence and decrease in rate of

115 A : PGA

0 :D HAP

C" RBP

Ch~r~hV. f l ~ e

T ffl

lmin L J

,-r

"°'°'",

I

0'05

l

.~

9-amino Icri~line fluorescence

/Jmoles 02

On

... . . . . . . . . . . . . . .

t

On

t

On

Figure 4. The effect of 2 mM glycerate-3-P (A), 25 gM DHAP and 200 ~M ribose-5-P (C) on chlorophyll fluorescence (top), 9-aminoacridine fluorescence (middle) and oxygen evolution (bottom). Solid lines are control traces with no added metabolite. Maximum rates of O 5 evolution are 26, 23 and 22.moles O~/mg chl/h in the controls for A, B and C respectively. Conditions as in Figure 1C. oxygen evolution both lag behind. Changes in both qE and qQ acting in parallel cause the changes in chlorophyll fluorescence yield. A similar result was obtained using protoplasts [ 11 ]. The broadly parallel, though phase shifted, relationship between ApH and chlorophyll fluorescence changes are consistent with the view that the rate o f oxygen evolution is seen to be inhibited when the ApH decreases and

116 increased when ApH is raised. Extensive studies using the reconstituted chloroplast system provide an explanation of this relationship. Using this system, it has been shown that the rate of glycerate-3-P dependent 02 evolution is very sensitive to changes in the ATP/ADP ratio [8, 13]. Phosphorylation of Rbu-5-P by Rbu.5-P kinase acts as an ATP sink which can therefore inhibit reduction of glycerate-3-P and consequent 02 evolution [1]. This competition between Rbu-5-P kinase and glycerate-3-P kinase for ATP therefore provides a mechanism for generating oscillation since both these enzymatic steps are required for sustained CO2-dependent 02 evolution [15, 18, 19] ; an 'excessive' production of Rbu-5-P caused by a high rate of glycerate-3-P reduction would suppress the subsequent rate of reduction and a series of adjustments would follow until rates of ATP consumption and production come into balance. Significantly, the addition of ribose-5-P (which is readily converted to Rbu-5-P) to the reconsistituted system elicits the exact sequence of events seen in Figure 1, with changes in chlorophyll fluorescence and rate of oxygen evolution lagging behind the primary change in 9-aminoacridine fluorescence [3, 5, 6]. This rationale is also consistent with the observed transient in the level of glycerate-3-P during a similar oscillation in protoplasts [12]. The effects of added metabolites are generally consistent with this rationale. Ribose-5-P lowers the ApH and suppresses the transient. Dihydroxyacetone.P would directly inhibit reduction of glycerate-3-P by mass action and again suppress the transient. With glycerate-3-P present the transient is again diminished since an enhanced glycerate-3-P level would decrease the inhibitory effect of low ATP/ADP. In this case, the transient was brought forward, indicating that the external addition of glycerate-3-P caused a more rapid generation of internal ribose-5-P. In all cases, the supply of external metabolites would interfere with the direct connection between the fluxes through glycerate-3.P kinase and Rbu-5-P kinase that is the proposed cause of imbalance and oscillation. Conversely, the appearance of the transient at low external Pi concentration indicates that here the connection between the metabolite fluxes has been made tighter. Hence, suppression of the export of triose phosphate by low Pi would mean that all the products of glycerate-3-P reduction would reach the Rbu-5-P kinase and enhance the potential for imbalance; export from the chloroplast would, in effect, buffer the stromal metabolite pools against abrupt change and so dampen oscillatory behaviour. In an accompanying paper evidence to support this view can be found in the enhancement of transient behaviour by inhibition of the phosphate translocator using pyridoxal phosphate [9].

Acknowledgements We wish to thank David Walker for his guidance and encouragement

117

throughout the course of this work. We are also grateful to many members of and visitors to the Institute for discussions on many aspects of this work but especially to Christine Foyer, Bob Furbank, Ulrich Heber, Richard Leegood, Hitoshi Nakamoto, Paul Quick and Mirta Sivak. Katherine Carver was a recipient of a S.E.R.C. studentship. This work was supported by A.F.R.C. References 1. Carver KA, Hope AB and Walker DA (1983) Adenine nucleotide status, phosphoglycerate reduction and photosynthetic phosphorylation in a reconstituted chloroplast system. Biochem J 2 1 0 : 2 7 3 - 2 7 6 2. Cerovic ZG, Sivak MN and Walker DA (1984) Slow secondary fluorescence kinetics associated with the onset of photosynthetic carbon assimilation in intact isolated chloroplasts. Proc Roy Soc Lond. B 2 2 0 : 3 2 7 - 3 3 8 3. Horton P (1983) Relations between electron transport and carbon assimilation simultaneous measurement of chlorophyll fluorescence, transthylakoid pH gradient and 02 evolution in isolated chloroplasts. Proc Roy Soc Lond B 217:405-416. 4. Horton P (1985a) Regulation of photochemistry and its interaction with carbon metabolism. In: Jeffcoat B, Hawkins AF, Stead AD (eds) Regulation of sources and sinks in crop plants. British Plant Growth Regulator Group, Bristol, pp 19-33. 5. Horto~ P (1985b) Interactions between electron transfer and carbon assimilation. In: Barber J, Baker NR (eds) Photosynthetic mechanisms and the environment. Elsevier Science Pubs, BV, pp 135-187. 6. Horton P, Lee P and Anderson S (1984) Fluorescence induction in a thylakoid system reconstituted for photosynthetic carbon assimilation. In:(Sybesma C ed) Advances in photosynthesis research Martinus Nijhoff/Dr W Junk Pubs, The Hague, Vol III, pp 6 5 7 - 6 6 0 7. Krause GH, Vernotte C and Briantais JM (1982) Photoreduced quenching of chlorophyll fluorescence in intact chloroplasts and algae, resolution into two components. Bioehim Biophys Acta 679:116-124. 8. Lilley RMcC and Walker DA (1974) The reduction of 3-phosphoglycerate by reconstituted chloroplasts and by chloroplast extracts. Biochim Biophys Acta 368: 269-278. 9. Nakamoto H, Sivak MN and Walker DA (1987) Sudden changes in the rate of photosynthetic oxygen evolution and chlorophyll fluorescence in intact isolated chloroplasts: the role of orthophosphate. Photosynth Res 11 : 119-130. 10. Quick P and Horton P (1984a) Studies on the induction of chlorophyll fluorescence in barley protoplasts. I. Factors affecting the observation of oscillations in the yield of chlorophyll fluorescence and the rate of oxygen evolution. Proc Roy Soc Lond B 220:361-370. 11. Quick P and Horton P (1984b) Studies on the induction of chlorophyll fluorescence in barley protoplasts. II. Resolution of fluorescence quenching by redox state and the transthylakoid pH gradient. Proc Roy Soc Lond B 220:271-382. 12. Quick P and Horton P (1986) Studies on the induction of chlorophyll fluorescence in barley protoplasts. III. Correlation between changes in the level of glycerate 3-phosphate and the pattern of fluorescence quenching. Biochim Biophys Acta, 849:1-6. 13. Robinson SP and Walker DA (1979) The control of 3-phosphoglycerate reduction in isolated chloroplasts by the concentrations of ATP, ADP and 3-phosphoglycerate. Biochim Biophys Acta 5 4 5 : 5 2 8 - 5 3 6 14. Sivak MN and Walker DA (1983) Some effects of CO 2 concentration and decreased O 2 concentration on induction fluorescence in leaves. Proc Roy Soc Lond B 217: 377-392. 15. Sivak MN and Walker DA (1985) Chlorophyll a fluorescence; can it shed light on fundamental questions in photosynthetic carbon dioxide fixation? Plant Cell and Environment 8 : 4 3 9 - 4 4 8

118 16. Sivak MN, Dietz K4, Heber U and Walker DA (1985) The relationship between light-scattering and chlorophyll a fluorescence during oscillations in photosynthetic carbon assimilation. Arch Biochem Biophys 237:513-519 17. Walker DA (1980) Preparation of higher plant chloroplasts. In: (San Pietro A ed) Methods in Enzymology, Academic Press, New York, Vo169, pp 94-104 18. Walker DA (1981a) Photosynthetic induction. In: (Akoyonoglou G ed) Proc 5th Int Cong Photosynthesis, Kassandra-Halkidiki, Balaban Int Sci Services, Philadelphia Vol IV, pp 189-202 19. Walker DA (1981b) Secondary fluorescence kinetics of spinach leaves in relation to the onset of photosynthetic carbon assimilation. Planta 153: 273-278 20. Walker DA and Sivak MN (1985) Can phosphate limit photosynthetic carbon assimilation in vivo? Physiologie Veg. Special Issue 23:829-841 21. Walker DA, Horton P, Sivak MN and Quick WP (1983) Anti-parallel relationship between 02 evolution and slow fluorescence induction kinetics. Photobiochem and Photobiophys 5: 35-39 22. Walker DA, Sivak MN, Ptinsley RT and Cheesbrough JK (1983) Simultaneous measurement of oscillations in oxygen evolution and chlorophyll a fluorescence in leaf pieces. Plant Physio173:542-549

Observation and characterisation of a transient in the yield of chlorophyll fluorescence in intact spinach chloroplasts.

A transient in chlorophyll fluorescence, which is associated with a transient in 9-aminoacridine fluorescence and a perturbation in the rate of oxygen...
428KB Sizes 2 Downloads 0 Views