PhowsynthesisResearch 50: 243-256, 1996. (~) 1996KluwerAcademicPublishers. Printedin the Netherlands. Regular paper

Cobalt induced changes in photosystem activity in Synechocystis PCC 6803: Alterations in energy distribution and stoichiometry Swati Tiwari & Prasanna Mohanty* School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India; *Author for correspondence Received 12 April 1996;acceptedin revisedform 14 November1996

Key words: cyanobacteria, electron transport, fluorescence, photosynthesis, spillover, state changes

Abstract

Adaptive responses to excess (supraoptimal) level of cobalt supplied to the growth medium were studied in the cyanobacterium Synechocystis PCC 6803. Growth of cells in the medium containing 10 #M COC12 led to a large stimulation (50%) in O2-evolution and an overall increase (,,~30%) in the photosynthetic electron transport rates. Analysis of variable Chl a fluorescence yield of PS II and immuno-detection of Photosystem II (PS II) reaction-center protein D1, showed a small increase (15-20%) in the number of PS II units in cobalt-grown cells. Cobalt-grown cells, therefore, had a slightly elevated PS II/PS I ratio compared to control. We observed alteration in the extent of energy distribution between the two photosystems in the cobalt grown cells. Energy was preferentially distributed in favour of PS II accompanied by a reduction in the extent of energy transfer from PS II to P S I in cobalt-grown cells. These cells also showed a smaller P S I absorption cross-section and a smaller size of intersystem electron pool than the control cells. Thus, our results suggest that supplementation of 10/~M COC12, to the normal growth medium causes multiple changes involving small increase in PS II to PS I ratio, enhanced funneling of energy to PS II and an increase in P S I electron transport, decrease P S I cross section and reduction in intersystem pool size. The cumulative effects of these alterations cause stimulation in electron transport and 02 evolution.

Abbreviations: BCIP-5-bromo-4-chloro-3-indolylphosphate; Chl a-Chlorophyll a; Cyt b/f-Cytochrome b/f; DCBQ-2,6-dichlorobenzoquinone; DCMU-3-(3,4-dichlorophenyl)-l,l-dimethyl urea; DCPIP-2,6-dichlorophenol indophenol; DPC-Diphenyl carbazide: Fo-ftuorescence when all reaction centers are open; FMfluorescence yield when all reaction centers are closed; Fv-variable chlorophyll fluorescence; HEPES-N-2hydroxyethyl piperazine-N'-2-ethanesulphonic acid; M V - methyl viologen; NBT-nitro-blue tetrazolium: p B Q para-benzoquinone; PBsomes- phycobilisomes; P C - Phycocyanin; P Q - plastoquinone; PS I - Photosystem I; PS II - Photosystem II; P 7 0 0 - reaction center Chl a of PS 1, ST - and MT-flash- single turnover and multiple turnover flash Introduction

An important characteristic of the photosynthetic apparatus of both prokaryotes and eukaryotes is its adaptability to everchanging and challenging environment. This is made possible by a concerted action and regulation of transcriptional, translational and bioenergetic processes which ensure efficient utilization of the light energy at a minimum cost. Such adaptive responses

may involve changes in gene expression and protein levels resulting in stoichiometric changes, and/or may also be achieved by changes in the fraction of light energy absorbed by a particular photosystem. Adaptations to environmental and nutritional alterations in cyanobacteria involves both short term and long term changes. Short term adaptation relates to change in the energy distribution between the two interacting photosystems. Unlike the case in higher

244 plants, the mechanism for regulation in distribution of excitation energy from phycobilisomes (PBsomes) to PS II and PS I, the phenomenon called 'state transition' has not been fully elucidated in cyanobacteria. It has been proposed that besides the redox state of PQ and/or Cyt b6/f complex (Allen 1992), the membrane energization generated by cyclic electron flow influence the state shifts (Fujita 1996). The long term adaptive changes relates to change in the antenna size, number of PBsomes feeding excitation energy to PS II, and via state changes to P S I as well as change in the stoichiometry of PS II to PS 1 (Allen 1992). In cyanobacteria, stoichiometry changes in response to light quality and quantity is a well- known phenomenon (Fujita 1996). Such adaptive changes involving changes in PS II to PS 1 ratio and intersystem electron transport chain not only alters photosynthetic electron flow but also influences the respiratory activities in cyanobacteria as in cyanobacteria both photosynthetic and respiratory electron transport chain reside in the same thylakoid membranes and share the same PQ pool (Bennoun 1982; Koike and Satoh 1996). Thus, in cyanobacteria, subtle changes in the environmental and nutritional conditions may induce both short term and long term adaptive changes. These changes alter the performance of photosystems and intersystem electron transport activity. From the above discussion, it is clear that the mechanisms responsible for the stress-induced damage to the photosynthetic apparatus and the corresponding adaptive responses is area of current research. In this context, we note that despite being detrimental for plant productivity and the environment, the mechanism of action of many metals at elevated concentrations on the adaptive responses of the photosynthetic organisms to metal toxicity are not well understood. Most knowledge related to metal toxicity is derived from studies of organelles. Such in vitro studies involve use of very high concentrations of metals which are not observed in realistic natural systems. The conclusions drawn from in vitro studies, therefore, can not always be extrapolated to in vivo situations. On the other hand, in vivo studies with metals are usually complicated due to interaction of the metal ions with the SH-groups of proteins, multiple sites of inhibition by the metal ions and ionic imbalances due to permeability changes caused by metal ions. The present investigation is directed towards understanding the adaptive mechanisms in Synechocystis PCC 6803 when grown in excess, but non-lethal (supraoptimal) levels of the essential micronutrient,

cobalt. Although specific uptake systems do exist for micronutrients, nonspecific transporters can take up excess micronutrient when it is present in higher than optimal concentrations in the growth medium and may either be accumulated inside the cells or actively excluded again (Nies and Silver 1989). Cobalt was selected for the present study due to various reasons given below. In vitro experiments with barley chloroplasts had shown that cobalt is not photooxidized by PS II (Hoganson et al. 1991). It inhibits the photosynthetic electron transport at PS II reaction center and has no effect on PSI (Tripathy et al. 1983). Unlike many other metal ions, cobalt ions do not have a strong affinity for SH-groups (Van Assche and Clijsters 1990). Therefore, secondary effects due to such interactions of the metal ions with proteins, if any, may be significantly minimized. Cobalt has been shown to be sequestered into polyphosphate bodies without causing any efflux or influx of other elements in the cyanobacterium Plectonema boryanum (Jensen et al. 1982). Considering that the effect of other metals in causing efflux or influx of other cations is similar in evolutionary distant organisms, possibly in Synechocystis PCC 6803 also cobalt may not upset the ionic balance of the cells. Unlike higher plants, cyanobacteria can adapt to changes in the nutritional status and other environmental changes quite readily (Reuter and Muller 1993). Our earlier studies have shown that growth of Synechocystis PCC 6803 cells in 10 #M COC12 stimulates the PS II electron transport rates (Tiwari and Mohanty 1993). In this communication, we have further characterized the nature of this stimulation in the electron transport activity. Since the ability to partition the excitation energy between the two photosystems is very sensitive to environmental conditions and nutritional status and the effect of metal ions on these processes is not very well understood, we have also evaluated the effects of supraoptimal concentration of cobalt on energy distribution between the two photosystems and their stoichiometry.

Materials and methods

Maintenance of cultures Axenic cultures of Synechocystis sp. PCC 6803 were maintained according to Rippka et al. (1979) in BG- 11 medium at 30 4- 2°C and illuminated with white fluorescent light at an intensity of 28-30 #E m -2 s -1. The

245 cultures were continuously bubbled with humidified filtered (Millipore, pore size 0.45 #m) air. For growing the cells at elevated levels of cobalt, four days old cultures were harvested and resuspended in BG11 medium at 5 #g Chl m1-1. COC12, (Sigma, tissue culture grade) was added to a final concentration of 10 #M. The growth curves of control and Co-grown cells did not show any appreciable differences. The cells were harvested in the exponential phase of growth, washed once and suspended in the required buffer for experiments. Estimation of cobalt contents in the cells by atomic absorption spectroscopy of thoroughly washed cells showed 0.02 and 2.09 #g cobalt g-1 dry weight of control and Co-grown cells, respectively.

Preparation of thylakoid membranes Thylakoid membranes were prepared according to Nilsson et al. (1992) with some modifications. The cells were harvested in the exponential phase of growth by centrifuging at 6000 x g for 5 min at room temperature and washed with buffer A [50 mM HEPES, pH 6.5, 10 mM MgCI2, 30 mM CaClz, 1 M sucrose and 25% (v/v) glycerol]. Cells were suspended in the same buffer containing 1 mM PMSF, at 0.2 mg Chl m l - i and subjected to 5 disruptions of 15 s each with 4 rain cooling intervals between the bursts in a bead-beater (Biospec Products Inc., USA). The homogenate was centrifuged at 6000 x g for 7 min at 4 °C. The pellet was washed once with buffer A, centrifuged and the two supernatants were pooled and centrifuged again at 30 000 x g for 45 min. The pellet thus obtained was suspended in buffer B (50 mM HEPES, pH 6.5, 10 mM MgCI2, 30 mM CaC12, 0.2 M sucrose and 25%, (v/v) glycerol] and was stored at - 8 0 °C. This was a crude membrane preparation and contained some phycobilins. The extent of contamination with phycobilins was around 35-40% as determined from the relative absorbance of Chl and phycocyanin (PC).

Electron transport measurements Electron transport rates were measured using a Clark type oxygen electrode (Hansatech, UK). For measuring PS II electron transport rates 1 mM pBQ or 100 #M DCBQ was used as an electron acceptor. The assay mix ( 1 ml) contained 50 mM HEPES, pH 7.5; 10 mM NaC1 and cells containing 5 #g Chl. P S I and whole chain electron transport rates were measured in thylakoids in the assay buffer described above. P S I electron trans-

port rates were measured by monitoring the oxidation of DCPIPH2 by MV in the presence of 10 #M DCMU and 5 mM sodium azide. Whole chain electron transport rates in the thylakoids were measured using DPC as an electron donor and MV as electron acceptor. Photosynthesis in intact cells involving electron transport in from water to CO2 was measured by adding sodium bicarbonate (10 mM) to the assay-mix. All measurements were done at 25 4- 1 ° C under saturating intensity of white light (1100 #E, m - 2 s - 1). Chl was estimated according to the method of Mackinney (1941 ).

Estimation of intersystem pool size Information about the intersystem electron transport chain, was derived by measuring P700 + redox kinetics using the pulse modulation system (PAM, Walz, Germany) fitted with an emitter-detector unit (ED 800T) which monitors the absorbance changes around 830 nm (Schreiber et al. 1988) arising due to P700 + radical at 810-830 nm. Measurements were done as described by Mi et al. (1992a, b). Cells were harvested and suspended at 100 #g Chl m1-1 in BG-11 medium. P700 was oxidized by application of far-red light (approx. intensity 100 #E m -2 s -1) for 10 s before turning the measuring light on. Single (ST) and multiple turnover (MT) saturating flashes of 6 #s and 50 ms durations, respectively, were applied with xenon discharge lamps XST-103 and XMT-103, respectively. In cyanobacteria, the respiratory electron flow and the cyclic electron flow around PS I are coupled with the intersystem electron chain (Bennoun 1982; Wollman and Delepelaire 1984; Scherer et al. 1988). To compare the contribution of these processes towards the overall P700-redox kinetics, measurements were also carried out in darkstarved cells depleted of respiratory substrates, and also after addition of HgCI2 (150 #M) to inhibit respiratory and cyclic electron flow (Mi et al. 1992a, b). For dark-starvation, the cells were incubated in the dark at room temperature (about 22 °C) for 32 h on a rotary shaker. Intersystem pool size was determined from the reduction areas obtained after single turnover (ST-) and multiple turnover (MT-) flashes.

State changes Cells were dark adapted for 2-3 h with mild shaking. Such dark adapted cells were considered to be in state 2 (Satoh and Fork 1983). For adaptation to state 1, dark adapted cells were illuminated with 150 #E m -2 s - I of far-red light obtained through a 711 nm inter-

246 ference filter (half band-width of 21 nm, Baird Atomic Inc., USA), for 5 min. Samples were frozen rapidly by dipping in liquid nitrogen. Subsequently, the sampies were held on a high purity copper finger dipped in liquid nitrogen (Shubin et al. 1991). The extent of state transition was monitored by recording fluorescence emission spectra on a Perkin-Elmer (model LS-5) spectrofluorimeter. The wavelength for excitation was 440 nm. Light intensity was measured with a LI-COR light meter (model LI- 189, LI-COR, USA).

Measurement of variable Chl afluorescence yield Variable Chl a fluorescence yield was measured as described by Philbrick et al. (1991 ) using PAM chlorophyll fluorimeter. Cells were washed and suspended at 20 #g Chl m1-1 in 50 mM HEPES, pH 7.5 containing 10 mM NaC1. The cell suspension was incubated for 5 min in dark in presence of pBQ (0.3 mM) and ferricyanide (0.3 mM), and Fo was measured. The intensity of modulated beam was kept low to avoid any actinic effect of measuring light itself. This was ensured, in a separate assay, by varying the measuring light intensity and measuring Fo in the absence and presence of DCBQ. An optimum setting was that which gave same value of Fo both in the absence and presence of DCBQ. Fo measurement was followed by addition of DCMU (10 #M) and NH2OH (20 mM) and further incubation for 30 s. Subsequently, FM was recorded by giving a saturating white light pulse (700 ms) from PAM-103.

Spillover measurements Spillover of excitation energy from PS II to P S I was measured from the interdependence of rise of fluorescence intensity at 685 (PS II fluorescence) and 725 nm (PSI fluorescence), with time of illumination at 77 K essentially as described by Srivastava et al. (1994). Cells were washed and suspended in BG-11 medium at 25 #g Chl m1-1. The fluorescence transients were recorded on a Perkin-Elmer spectrofluorimeter (model LS-5). Ceils were adapted to state 1 or state 2 at room temperature as described above and rapidly frozen in liquid nitrogen. Fluorescence was excited by 440 nm light (excitation slit = 15 nm) of low intensity (0,12 #E m -2 s- l) obtained by using a blue band pass filter (Coming CS 4-96). The kinetics of fluorescence rise was monitored at either 685 nm (for PS II) or 725 nm (for PS I). The emission slit was kept at 10 nm. An average of Fv/FM at various times of induction from many identical samples was taken and the normalized

variable fluorescence (Fv/FM) from PS I was plotted as a function of normalized variable fluorescence from PS II.

Absorption cross-section of PS I The absorption cross-section of P S I was estimated by measuring the kinetics of photooxidation of P700 manifested as a decrease in absorbance at 700 nm on illumination of thylakoid membranes (Melis and Andersson 1983). The measurements were carried out on a modified Hitachi UV-220 spectrophotometer that provided actinic light to the sample cuvette while keeping the reference cuvette in dark. Thylakoid, were suspended in 50 mM HEPES, pH 7.5 containing 10 mM NaC1 to a concentration of 40 #g Chl ml -~ . The assay mix also contained 10 #M DCMU, 5 mM Na-ascorbate, 0.5 #M DCPIP and 200 #M MV. Oxidation of P700 was initiated by illumination with a blue light of limiting intensity (25-30 #E m -2 s- 1) obtained by passing white light through a Corning CS 4-96 filter. Semilogarithmic plot of the decrease in absorbance at 700 nm (due to oxidation of P700) against time is a straight line, the slope of which indicates the functional antenna size of PS I.

Immuno-blot analysis Thylakoid membrane proteins were solubilized in 6 M urea, 10% (w/v) SDS, 2.5% (v/v) 2-mercaptoethanol, 0.125 M Tris-HC1, pH 6.8 and separated on a 12.5% (T) SDS-acrylamide gel containing 2 M urea. Gels were washed after electrophoresis and blotted on nitrocellulose membranes (0.45 #m, Millipore). Blots were probed with antibodies against DI and psaA/B, that were raised in rabbit (antibodies against D1 were a kind gift of Dr H. Pakrasi and those against psaA/b of J. Enami). Blots were incubated with anti-rabbit IgG antibodies coupled to alkaline phosphatase and developed using NBT/BCIP as substrate.

Results and discussion

Our previous results had shown that the growth of

Synechocystis PCC 6803 cells in the presence of 10 #M cobalt chloride stimulates the PS II electron transport rates by > 50%, (Tiwari and Mohanty 1993). Other essential micronutrients namely Zn, Mn, B, Cu and Ni when added to 10 #M concentration in the BG-11 medium inhibited pBQ supported PS II catalyzed Hill

247 300 -

---o---

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CGNT~ Co

250 Mn

200

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,~

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Mo

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-

150 -

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50 ¸

I

I

I

I

I

i

10

20

30

40

50

60

TIME

(h)

Figure 1. Effect of sublethal concentrations of selected micronutrients on pBQ-supported PS II electron transport rates at different time points of growth. Each time point represents an average of two independent experimental sets. The standard deviation was in the range of 6.1 to 14.1 with the maximum standard deviation observed with Cu-growth cells. Table 1. Electron transport rates in intact cells and thylakoids of Synechocystis PCC 6803 cells grown in 10/zM COC12 Material

Assay

Control

Co-grown

% Change

H20 --+ pBQ

318.0

455

43

H20 --+ DCBQ H20 --+ CO2

228.0 31.0

342.0 41.0

50 29

68.3 H20 --+ DCBQ DCPIP --->MV 41.0

91.1 98.0 285.0 52.0

33 130.0 341.0 27

63 27

Intact cells

H20 --->pBQ Thylakoids DPC --+ MV

Assay mix contained 5 #g Chl ml-[ equivalent cells or thylakoids in 50 mM HEPES, pH 7.5, 30 mM CaC12, 10 mM NaCI, 5 mM MgCI2. The final concentrations of pBQ and DCBQ were 1 mM and 0.5 mM, respectively. The assay mix for DCPIP to MV electron transport contained 5 mM sodium ascorbate, 100/zM DCPIP, 5 mM sodium azide, 10 #M DCMU and 50/zM MV. For whole chain electron transport measurement in thylakoids, DPC was 0.5 mM and MV was 50/zM. H20 to CO2 rates were measured in intact cells with 10 mM sodium bicarbonate in the assay buffer. Rates are in/~mol 02 (mg Chl)- [ h - l and are an average of at least two experimental sets.

r e a c t i o n to v a r y i n g d e g r e e s ( F i g u r e 1). I n c r e a s i n g the c o n c e n t r a t i o n o f COC12, to 2 0 / z M also r e s u l t e d in an i n h i b i t i o n o f e l e c t r o n t r a n s p o r t rates ( d a t a n o t s h o w n ) . T h i s e f f e c t o f c o b a l t s t i m u l a t i o n o f 0 2 e v o l u t i o n at 1 0 / z M a n d i n h i b i t i o n at m u c h h i g h e r c o n c e n t r a t i o n s is r e m i n i s c e n t o f t h e e f f e c t o f s o m e h e r b i c i d e s w h i c h also c a u s e a s t i m u l a t i o n o f PS II e l e c t r o n t r a n s p o r t at

s u b l e t h a l l e v e l s ( H e r c z e g et al. 1980) b u t are i n h i b i t o r y at h i g h e r doses. E l e c t r o n t r a n s p o r t rates PS II e l e c t r o n t r a n s p o r t rates s h o w e d 4 3 % h i g h e r rates w i t h p B Q a n d 5 0 % s t i m u l a t i o n w i t h D C B Q as e l e c t r o n

248

TIME (s)

Table 2. The effect of 10/zM COC12 on the absorption cross section of P S I in Synechocystis PCC 6803

0

thylakoids

Set Set 2 Set 3

0 _8

Control

KI Co-grown

% Change

1.30 + 0.12 1.82 -4- 0.20 1.85 -4- 0.16

0.95 + 0.05 1.42 + 0.13 1.36 4- 0.11

28.6 22.0 26.5

Kl values are an average from the slope of three kinetic plots recorded for the samples drawn from the same thylakoid preparations. Kj is very sensitive to the light intensity used for the photooxidation. Hence, the values for each set are given separately.

.5

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acceptors in the Co-grown cells compared to control on an equal chlorophyll (Chl) basis (Table 1). The stimulation in the DCBQ supported PS II rates was 63% in the thylakoids prepared from the Co-grown cells, though the rates were much lower than those obtained with intact cells (Table 1). Thylakoids from the Co-grown cells also had a 27% higher activity of the whole chain electron transport (Table 1) as observed from DPC to MV assay. Since DPC donation site to PS II bypasses the water oxidase system (Vernon and Shaw 1969; Hsu et al. 1987), it appears that this stimulation of electron transport rates in the Co-grown cells not only involves the water oxidase system but also the entire electron transport chain. Also, the extent of this stimulation in the Co-grown cells was the same as that of the physiological electron transport rates measured in intact cells with CO2 as acceptor (Table 1). P S I electron transport rates were also stimulated but by about 20% only in thylakoids from the Co-grown cells at saturating intensity of white light (Table 1).

Figure 2. A typical semi-logarithmic plot of the kinetics of P700 photooxidation in the thylakoids of Synechocystis 6803.20 #g Chl equivalent thylakoids were taken for each assay. Each point represents an average of four values. Values of KI determined from the slope are given in Table 2 for three separate experiments. The standard error at each point is less than 5%.

vidual experiments since KI is extremely sensitive to changes in light intensity. A decrease in K1 indicated that Co-grown cells had a smaller PSI absorption cross section compared to control. However, the reduction observed in the absorption cross section of Co-grown cells reflects the functional ability and does not necessarily reflect structural organization. However, an increase in the P S I electron transport rates in the Cogrown cells despite having a 30% smaller PSI absorption cross-section suggests that there must be other structural-functional changes in the electron transport chain in response to supraoptimal levels of cobalt.

lntersystem pool-size PS I absorption cross-section To ascertain if the enhancement in P S I was accompanied by changes in the P S I antenna, the relative antenna size of P S I was determined in thylakoids of control and Co-grown cells. An estimate of the rate of light absorption by PSI was obtained from the slope K1 of the semi-logarithmic plots of the kinetics of P700 photooxidation (Figure 2). K] is proportional to the size of PS I antenna at a given intensity. The KI for thylakoids isolated from Co-grown cells was found to be 23 to 30% lower compared to the control (Table 2). The variation between different experimental sets could be due to small changes in the actinic light intensity used to induce photooxidation of P700 in indi-

Of the possible mechanisms that could lead to an increase in the PSI rates is an alteration in the redox status of PS I. Thus, we have studied the electron donation from intersystem pool by monitoring the absorbance changes due to P700 + radical at 820 nm. P700 was oxidized with a continuous far-red (FR) light and the absorbance at 820 nm was zeroed to give a base-line. The saturating single turnover, (ST) flash causes a single charge separation event at Photosystem II and I, while the multiple turnover (MT) flash additionally causes electron flow between the two photosystems. Thus, the applications of ST- and MT-flashes in conjunction with far-red light that oxidizes P700 of PS I enables to estimate the intersystem pool size (Schreiber

249 Table 3. The intersystem carrier pool size of control and Cogrown cells

Treatment

AreaMT/AreasT % Change Control Co-grown

None 32 h dark adaptation 32 h dark adaptation + 100 #M HgC12

53.8 4- 2.0 96.8 4- 9.1

15.4 4- 2.7 69.2 -4-4.8

71.0 28.5

39.3 4- 2.5

35.04- 6.2

10.9

Cells were suspended at 90/~g Chl ml- l in BG-11 medium.The extentof P700oxidationwas measuredas absorbanceat 820-830 nm using PAM fluorimeter. 100/LMHgCI2was added in dark, 2 min priorto measurement.AreaMTand AreasTale the reduction areas after a single-and a multiple-turnover flash, respectively. Each valueis an averageof three independentmeasurements(for details see 'Materials and Methods').

et al. 1988). The extent and the rate of photooxidation of P700, the physiological donor to PS I, depends on the charge separation. The rate of re-reduction of P700 + is dependent on the charge separation and the redox state of intermediate electron pool which gets reduced by electron flow from PS II. Figure 3 shows the redox kinetics of P700 under far-red illumination and by additional ST- and MTsaturation pulses. A further oxidation of P700 in both control and Co-grown cells on additional application of ST- and MT-flashes (Figure 3A, B) is indicative of respiratory and/or cyclic electron flow through PS I, which is common in cyanobacteria (Bennoun 1982; Wollman and Delepelaire 1984; Scherer et al. 1988). In Cogrown cells the extent of P700 oxidation observed on additional application of ST- and MT-flashes was much larger than that in the control suggesting that Co-grown cells may have a significantly more cyclic and/or respiratory activity than the control cells. The fact that the re-oxidation of P700 after the multiple-turnover flash was much slower in the Co-grown cells compared to control (Figure 3A) also suggests that P700 is kept reduced by electrons either from the respiratory or from cyclic electron flow. In the absence of such an activity, a ST-flash in presence of background far red light leads to a complete oxidation of intersystem electron transport chain. Application of a MT-flash when P700 and the intersystem electron transport chain is almost fully oxidized, gives an almost complete re-reduction of P700 (observed as a decrease in absorbance at 830 nm (A830)) which is again re-oxidized by the background FR light observed as an increase in A830 to

baseline level. The time required to re-oxidize P700 will be proportional to the number of electrons stored in the PQ-pool. Therefore, a comparison of the reduction areas gives a measure of the intersystem pool size (Schreiber et al. 1988). The reduction area was 53 in control cells, but only 15 in Co-grown cells (Figure 3A, Table 3). Hence, the control cells appeared to have a minimal intersystem carrier pool capacity of 53 electron equivalents compared to only 15 to 16 electron equivalents in the Co-grown cells. However, this does not reflect a correct estimate of the intersystem pool because of the recycling of electrons from cyclic and respiratory flow around PS I. Therefore, the cells were incubated in dark for 32 h (dark starvation) to deplete the respiratory substrates (Mi et al. 1992a, b). In these cells donation of electrons to the intersystem pool will be only from linear and cyclic flow around PS I. Determination of P700 redox kinetics in these cells showed the intersystem pool size to be 97 in control and 69 in Co-grown cells (Figure 3B, Table 3). These higher values compared to those observed for Figure 3A could be due to an increase in the cyclic flow around P S I to maintain a higher ATP/NADH ratio in these cells depleted of respiratory substrate. Addition of HgCI2 to the dark starved cells prior to measurement, to inhibit electron donation from NAD(P)H to the plastoquinone pool, is likely to give a correct estimate of the intersystem pool size because in this situation the only source of electrons into the intersystem pool will be the linear electron transport (Mi et al. 1992b). Under these conditions, the intersystem pool size was 39.3 and 35 for control and Co-;grown cells, respectively (Figure 3C, Table 3). Thus the Co-grown cells seemed to have an 11% smaller intersystem pool size. The absorbance change after the MT-flash was almost the same for both control and Co-grown cells (Figure 3C) which suggests that there may nut be any appreciable change in the P700 content of the Co-grown cells. Although the number of P S I units appeared to be unchanged according to the abovementioned experiment, and there was about 30% reduction in the functional absorption cross-section of P S I due to the Cotreatment, there was about 20% increase in the PS I electron transport rates in the thylakoids from Cogrown cells. Moreover, in spite of an estimated 11% decrease in the size of the intersystem electron pool, the overall electron transport rates were higher (about 20%) in the Co-grown cells (Table 1). A possible explanation for these intriguing observations could be an increase in the rate-constant of either PQ oxidation by the Cyt b6/f complex or reduction of plastocyanin by

250 ST

MT

ST

eAT

r 't- O O rt

N

Tr¢ci¢cd

Cofllror

TIME ( $ )

Figure 3. Determination of intersystem-poolsize in control and Co-grown cells by redox changes of P700 at 820 nm. Cells were suspended at I00 #g Chl m1-1 in BG-I1 medium and illuminated with far-red light (about 100/LE m -2 s -1) for 10 s and the absorbance at 820 nm was recorded using the emitter-detector unit (ED 800) of PAM fluorimeter. The absorbance was set to zero for baseline. Single (ST) and multiple (MT)-turnover saturating flashes were given as indicated in the figure. (A) redox changes in the freshly suspended cells without any dark incubation, (B) redox changes after 32 h dark incubation of the cells and (C) redox changes of the 32 h dark adapted cells in the presence of 150 /zM HgC12. HgCI2 was added in dark, 2 min prior to the measurement. The absorbance scale is the same in all the three cases.

Cyt f which is manifested in a faster kinetics of P700 re-oxidation in Co-grown cells (Figure 3C). The latter reaction occurs extremely rapidly with rate constants > 2 3 0 0 s - l (Haehnel et al. 1980) relative to the ca. 2 5 0 350 s - 1 rate constant for PQH2 oxidation by the Rieske center (Selak and Whitmarsh 1982). Since PQH2 oxidation step is considered to be the rate-limiting step of linear electron transport (Witt 1971), the stimulation observed in the electron transport rates may be due to an increase in the rate constant o f PQH2 oxidation by the Rieske center.

Alterations in state shift ability Adaptive responses o f the cells to excess but nonlethal (supraoptimal) levels of cobalt were also studied in terms of the changes in the relative partitioning of light energy by the two photosystems (state changes)

which is manifested as changes in the fluorescence emission spectra at 77 K (Murata 1969; Mohanty et al. 1973). Figure 4 shows the emission spectra at 77 K of intact control and Co-grown cells adapted to either state 1 or 2. The excitation wavelength was 440 nm, which excites mainly Chl a. Both the control and the Co-grown cells exhibited the characteristic emission peaks for PS II (685 and 695 nm) and PS I (725 nm) though the relative intensities of the peaks were different in both the samples and between different light states (Figure 4). The small emission hump at 655 nm represents the emission due to PBsomes. In state 2 adapted cells, a higher proportion of energy is distributed in favour of PS I, conversely in state 1 adapted cells, energy is distributed in favour of PS II (Murata 1969; Mohanty et al. 1973). Hence, the ratio o f fluorescence intensity of PS I (F725) to PS II (F685), which reflects the relative distribution of excitation energy

251 Table 4. The relative ratio of F725:F685 fluorescence emission intensities of control and Co-grown Synechocystis 6803 cells recorded at 77 K Control

Co-grown

F725 :F685

% Change

F725 :F685

% Change

State 1

2.53 d- 0.06

48.0

2.55 -4- 0.02

11.5

State 2

3.755:0.15

2.884-0.23

The cells were suspended in BG-11 medium at 5 /zg Chl ml - I . The cells were adapted to either state 1 or state 2 and frozen rapidly in liquid nitrogen. The excitation wavelength was 440 nm. The excitation and emission slits were 10 and 5 nm respectively. The ratio of F725 :F685 were calculated after normalization at 655 nm and represent an average of three sets of experiments. % change denotes change in the ratio of F725:F685 in shifting from state 2 to state

I

u

c

/

c u o

600

1 I 650 ?00 Wovelength ( n m )

I'50

I

600

650 700 W o v ¢ l e n g t h (rim)

750

Figure 4. 77 K fluorescence emission spectra of intact Synechocystis 6803 cells in light state 1 and 2. Cells were suspended in BG-I 1 medium at 5 #g Chl m l - I. They were adapted to either state 1 or state 2 and rapidly frozen in liquid nitrogen. The excitation wavelength was 440 nm and the excitation and emission slit widths were 10 and 5 nm, respectively. (A) Control, (B); 10/~M COC12 grown cells. State 1, solid lines; state 2, dashed lines.

between the two photosystems, is higher in state 2 adapted cells (Murata 1969). Table 4 shows that the ratio of F725/F685 in Co-grown cells was about 23% lower than that of control cells in state 2 while it was almost equal in state 1. Thus, more energy seems to be partitioned in favour of PS II in Co-grown cells. Moreover, the increase in this ratio in shifting from state 1 to state 2, was 48% for control cells and only 11.5% for Co-grown cells. This suggested that the Co-grown cells had an altered capacity to undergo state transitions. It appears that state 2 adapted control cells can distribute relatively more energy in favour of P S I than

Co-grown cells in state 2 (Figure 4, Table 4)., In other words, Co-grown cells appeared to be preferentially shifted towards state 1. The kinetics of state change in going from state 2 to state 1 was, however, similar in both control and Co-grown cells (data not shown).

Changes in spillover The effect of COC12 in the growth medium on the transfer of excitation energy from PS II to P S I (spillover) was also evaluated. Since P S I fluorescence yield is not linked to the redox state of PS I, it does not have

252 a variable fluorescence (Kitajima and Butler 1975). In the cyanobacterium Spirulina, a small variable fluorescence has been reported (Shubin et al, 1991) but this variable yield in fluorescence has not been characterized in terms of quantitative energy distribution models. According to Butler's bipartite model of energy distribution between photosystems (Butler 1978), variable fluorescence (Fv) from P S I arises solely due to energy transfer from PS II. Therefore, the analysis of variable fluorescence from PS II and PS I may be utilized to derive information about the extent of energy transfer from PS II to PS I. Upon illumination of the frozen sample at 77 K, a rise in fluorescence intensity observed at 685 and 725 nm is attributed to fluorescence from PS II and PS I, respectively. A kinetic plot of P S I fluorescence emission versus PS I fluorescence emission obtained during fluorescence induction at 77 K shows a linear relationship (Kitajima and Butler 1975). While the slope of the curve may be taken to be proportional to the rate constant of spillover from PS II to PS I, the Y-intercept is proportional to the absorption cross section of P S I (Kitajima and Butler 1975). In our study, the fluorescence induction transients were recorded separately for emission from PS 1 and PS II. Since the induction kinetics was recorded for fluorescence from PS II and P S I using different samples, we have taken an average of Fv/FM at different time intervals of induction curve from many identical sampies. The normalized variable fluorescence (F~/FM) from PS I was plotted as a function of normalized variable fluorescence from PS II (Figure 5). The slope indicates the spillover. However, no information could be derived about the absorption cross section of P S I due to normalization (see 'Materials and methods'). We chose to normalize to FM and not to Fo because of the limitation in the instrument's response time which would have given an overestimate in the Fo. However, this normalization is not likely to affect the slope which was used to determine the extent of spillover in this study (Srivastava et al. 1994). Figure 5 shows that the slope was higher in state 2 as compared to that in state 1 in both control and Co-grown cells. An increase in the slope indicating an increase in the spillover of excitation energy is in agreement with decreased spillover in state 1 adapted cells of Porphyridium (Ley and Butler 1980), Anacystis nidulans (Bruce et al. 1985) and Synechocystis 27170 (Post et al. 1991). As shown in Table 5 each batch of cells, control or Co-grown, showed an increase in the slope accompanying transition to state

2. The extent of increase in the slope of control cells, when going from state 1 to state 2 was 35 to 58%. Cogrown cells, on the other hand showed only 2 to 7% increase in this ratio. Fluorescence emission spectra at 77 K. indicated that the Co-grown cells were shifted towards state 1 and had a reduced ability to shift to state 2 in terms of energy distribution between PS II and P S I (Figure 4, Table 4). A minor change in the slope (2 to 7%) on going from state 1 to state 2 (Figure 5) is in agreement with these results and suggests a considerable reduction in spillover even during state 2 condition in Co-grown cells. Partitioning of energy mostly in favour of PS II also explains the observed decrease in the functional P S I antenna of the Cogrown cells as determined from the kinetics of P700 photooxidation.

Fluorescence yield analysis We also determined the concentration of PS II centers in the cells to ascertain the relative contribution of the short- and the long-term changes in eliciting a meaningful adaptive response to the metal stress. The relative concentration of PS II centers was measured by comparing the variable fluorescence induced by saturating white light in the presence of hydroxylamine and DCMU (Philbrick et al. 1991). While DCMU inhibits electron transfer from QA to QB, hydroxylamine prevents charge recombination between P680 + and QAby keeping P680 in a reduced state. Thus, maximal PS II fluorescence is obtained under these conditions when actinic light is given. Table 6 shows that the variable fluorescence (Fv) of the Co-grown cells was about 14% higher than that of control cells. The Fo of Co-grown cells was also higher than that of control cells by about 14%. Since cells were incubated with pBQ and FeCN prior to measurement of Fo, the values obtained could be considered to be true reflection of Fo. The dependence of the fluorescence yield on [QA]-1 is non-linear when energy transfer occurs between centers, but linear in the absence of such energy transfer (Joliot and Joliot 1964). Since the latter situation appears to prevail in Synechocystis PCC 6803, a measurement of FM - Fo when all QA is reduced can give a fairly reliable measure of the relative PS II center concentration. A similar increase in Fo and Fv reflects a slight increase in the number of PS II centers in Co-grown cells. The same extent of increase in Fv and Fo values is in agreement with our previous result that there is no significant change in the absorption cross-section of PS II (Tiwari and Mohanty 1993).

253 LO0 -

'

1.00

It'} I,w

l.k,

0.75

0.75 -

'U.

050 0 50

1

0 75

050

i

OSO

1 O0

075

I O0

F 685

F 685

Figure 5. Yield of variable fluorescence due to P S I (F725) as a function of variable fluorescence due to PS II (F685) during fluorescence induction at 77 K. Cells were suspended in BG-I 1 medium at 25 #g Chl m l - l, adapted to state 1 or 2 and rapidly frozen in liquid nitrogen. The excitation wavelength was 440 nm and the excitation and emission slit widths were 10 and 5 nm, respectively. The yield of variable fluorescence was normalized to the maximal fluorescence. Typical plots from (A) control and (B) Co-grown cells are shown. State 1, closed circles; state 2, open circles. Table 5. Changes in spillover of excitation energy from PS II to PSI due to state changes in intact cells of Synechocystis 6803

Set 1 Set 2 Set 3

State 2

Control State 1

1.00 0.98 1.95

0.74 0.74 1.25

Change (%)

State 2

35.0 32.0 56.0

1.07 0.92 1.35

Co-grown State 1 Change (%) 1.00 0.90 1.30

7.0 2.2 3.8

The values represent the slope of the linear curve when Fv/FM from PSI is plotted as a function of Fv/FM from PS II during fluorescence induction at 77 K. Each value is an average of four transients. % change denotes change in spillover of excitation energy in shifting from state 2 to state 1. Table 6. Variable fluorescence yield of Chl a in control and Co-grown cells Control

Set 1

Co-grown

% Change

F,,

Fv

Fo

Fv

Fo

Fv

19.3 4- 0.02

17.5 4- 0.00

21.3 4- 0.45

20.3 4- 0.34

10.4

16.0

29.8 4- 0.06 21.5 4- 0.24

29.8 -4- 0.50 19.3 4- 0.43

14.6 14.9

17.8 17.7

1

Set 2 Set 3

26.0 4- 0.41 18.7 4- 0.01

25.3 4- 0.50 16.4 4- 0.01

Cells were suspended at 20 #g Chl m1-1 in 50 mM HEPES, pH 7.5, 10 mM NaCI and incubated with 0.3 mM each ofpBQ and ferricyanide for 5 min in dark and E, was measured. Relative yield of chlorophyll fluorescence (Fv = FM - Fo), was measured in the presence of 10 #M DCMU and 20 mM hydroxylamine, by giving saturating white light (700 ms). Each value represents an average of five measurements. The variation in the values is due to difference in the recorder setting between separate experiments.

T h e e x t e n t o f i n c r e a s e in t h e n u m b e r o f P S II u n i t s w a s also determined from the complementary area above t h e f l u o r e s c e n c e i n d u c t i o n c u r v e in t h e p r e s e n c e o f 5 # M D C M U . T h i s a r e a at a g i v e n t i m e is a s s u m e d to

b e p r o p o r t i o n a l to t h e f r a c t i o n o f r e d u c e d QA ( M e l i s a n d D u y s e n s 1970). C o - g r o w n c e l l s s h o w e d a 11 to 14% higher value of the complementary

area above

the induction curve (data not shown) suggesting an

254

Figure 6. Immuno-blotof thylakoid proteins from control and Cogrown cells. Lanes 1-3 show coomassieblue stainedgel. Lanes4--7 show immuno-blotswith anti-D1 and anti-psaA/B antibodies.Lane 1, molecularweight markers in kDa; lanes 2, 4 and 6 thylakoids fromcontrol cells; lanes 3, 5 and 7, thylakoidsfromCo-growncells. Lanes 4 and 5 are probed with anti-Dl antibody and lanes 6 and 7 with anti-psaA/B antibody.

increase in QA which may be indicative of a small increase in the number of PS II units in these cells.

Quantitation of reaction center proteins Change in the stoichiometry of PS II to PS I were confirmed by immuno-blot analysis of thylakoid membranes from control and Co-grown cells, using antibodies against D1 and psaA/B, which are reaction center proteins of PS II and PS I, respectively. Figure 6 (lanes 2 and 3) show coomassie blue stained polypeptide profile of thylakoids from control and Co-grown cells, respectively when run on equal chlorophyll basis. Figure 6 (lanes 4, 5) shows immuno-blot with anti-D1 antibody and lanes 6, 7 with anti-psaA/B antibody. Anti-D1 antibody gave two bands of apparent molecular weights of 32.4 and 30.2 kDa, This lower molecular weight band has been observed by Groloubinoff et al. (1988) and other workers also and is suggested to be a conformer of D1 or incompletely denatured D1. Densitometric analysis of blots from three different preparations showed 15-20% increase in D1 protein and no change in psaA/B protein levels in Co-grown cells. Therefore, we conclude that Co-grown cells had

a slightly higher ratio of PS II/PS 1 compared to control. There are several examples of a stimulation in the photosynthetic activity to adapt to environmental stress. For example, a stimulation in PS II activity has been observed when plants are grown in presence of sublethal doses of certain herbicides (Herczeg et al. 1980; Bose et al. 1984; Laski and Lahoczki 1986). A stimulation in the photosynthetic activity has also been reported in Nostoc muscorum (Blumwald and Tel-Or 1982), Synechococcus (Blumwald and TelOr 1984) and in Brassica (Alia et al. 1992) due to salt-stress. An increase in the photosynthetic activity has been correlated with a need to meet the energy demands of the cells for the production of soluble sugars and osmoregulation (Blumwald et al. 1983). Since cyanobacteria are known to accumulate sugars, glycerol, etc. under stress, a similar need for more energy and hence increased photosynthetic activity cannot be ruled out in Synechocystis cells grown in supraoptimal levels of cobalt chloride. Our present investigation demonstrates that the growth of Synechocystis PCC 6803 cells in moderately elevated (10 #M) levels of cobalt leads to a large stimulation in PS II electron transport rates. The stimulation of 02 evolution and electron transport rates was attributed to multiple effects induced by cobalt. When cobalt was present in excess of this supraoptimal, 10 #M level, it causes inhibition or reduction in 02 evolution similar to other metal cations. However, with small elevated (10 #M) level of cobalt, there was a small increase in the number of PS II reaction centers as evidenced from quantification of reaction centre proteins and fluorescence analysis. A small increase in the P S I electron transport rates was also observed but there was no change in the number of PS I units in the Co-grown cells. The Co-grown cells were shifted towards state 1 which favours a preferential energy distribution in favour of PS II. This was substantiated by our analysis of 'spillover' which showed that energy transfer from PS II to P S I was inhibited in Co-grown cells. We have also observed a change in the rate of feeding of PQH2 and Cyt b6/fto P S I donor. In summary, the growth of cells in moderately high levels of cobalt leads to higher photosynthetic rates in the Co-grown cells as manifested by 30% higher rates of whole chain electron transport. Also. there appears to be an increase in the number of active PS II centers in Co-grown cells. However, the extent of this increase was only about 15-20% and it does not account for the large (30-50%) stimulation observed in the photosyn-

255 thetic activity. This small increase in the PS II:PS I ratio was a c c o m p a n i e d by a preferential distribution o f energy in f a v o u r o f PS II and the s u m total o f these changes i n d u c e d by cobalt brings about the enhancem e n t in o x y g e n evolution. We note that the excess level or the supraoptimal cobalt concentration in the BG-11 m e d i u m used for g r o w i n g Synechocystis P C C 6803 has a dramatic effect on the photosynthetic ability of the cells particularly in energy distribution b e t w e e n the p h o t o s y s t e m s and other changes that affect the functional ability o f electron transport chain. W h e t h e r the o b s e r v e d a d a p t i v e responses are due to a direct interaction of cobalt with PS II or are a c o n s e q u e n c e o f an indirect effect is, h o w e v e r not clear at present.

Acknowledgements This w o r k was supported by the grant C S I R 0 9 / 0 3 7 4 / E M R I I . W e thank Dr M i k e Siebert ( N R E L C o l o r a d o , U S A ) , for the gift o f Synechocystis P C C 6803 and Dr N i c k B u k h o v , Institute o f Plant Physiology, M o s c o w for the gift o f optical filters. We also thank D r M. Ikeuchi, U n i v e r s i t y o f Tokyo, Dr I. Enami, S c i e n c e U n i v e r s i t y o f Tokyo, Japan, Dr A. Mattoo, U S D A , M a r y l a n d , U S A and P r o f H. Pakrasi, Washington University, St. Louis, U S A for the gifts o f antibodies. We express our thanks to Dr Kolli Bala Krishna for his help.

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