Does Cd2+ use Ca2+ channels to penetrate into chloroplasts? — a preliminary study

ACTA PHYSIOLOGIAE PLANTARUM Vol. 22. No. 2. 2000:171-178

D o e s C d 2+ u s e C a 2+ c h a n n e l s to p e n e t r a t e i n t o c h l o r o p l a s t s ? - a preliminary

study

Ewa Skdrzyfiska-Polit* and Tadeusz Baszyhski Department of Plant Physiology, Maria Curie-Sktodowska University, Lublin 20-033, Akademicka 19, Poland * For correspondence: e-mail: eskorzyn @biotop.umcs.lublin.pl

Key words: C a d m i u m , Calcium, Channel, Fluorescence, Ionophore, Phaseolus coccineus, P r i m a r y photochemistry, PSII, Quenching

Abstract Effects of Cd2+ toxicity on the photochemistry of primary leaves at two different growth stages of runner bean plants were taken into consideration to study whether C d 2+ c a n use Ca2+ channels to get into chloroplasts• Different concentrations of Cd2+, ionophore A 23187 and Ca2+ were vacuum infiltrated into leaf discs. Toxicity of Cd2+ at the donor side of PSII depending on the metal concentration and age of the plants was confirmed. Application of ionophore caused an increase in the sensitivity of the PSII donor side to low Cd2+ concentrations. Additional suppl12of Ca2+ in the infiltration medium abolished toxic effect of Cd on photochemical activity, excel2tfor older plants, where it was not observed for the highest Cd z+ concentratmn. In our oplmon it is possible that Cd penetrates mto chloroplasts via Ca2+ channels. Age-dependent Ca2+ content in the primary leaves seems to be a very important factor protecting photochemical activity from the toxic action of Cd2+ . •

.

,Z+

.

.

.

-

.

.

2+

List of abbreviations: CP43, CP47 - core proteins 4 3 k D a and 4 7 k D a o f p h o t o s y s t e m II; F m - m a x i m a l chlorophyll a fluorescence w h e n all PSII reaction centers are closed in dark-acclimated leaves; FO minimal chlorophyll a fluorescence when all P S I I reaction centers are open in darkness; Fv - variable chlorophyll a fluorescence; Fv/Fm - m a x i m a l p h o tochemical yield o f P S I I in dark-acclimated leaves;

L H C I I - light-harvesting chlorophyll a/b c o m p l e x o f PSII; L N U - light a b s o r b e d not used in primary p h o t o c h e m i s t r y ; P P F D - photosynthetic photon flux density; qN - n o n p h o t o c h e m i c a l quenching of fluorescence; qp - p h o t o c h e m i c a l quenching of fluorescence

Introduction C a l c i u m is an essential c o m p o n e n t o f plant cells. It plays m a n y important structural and physiological roles in plants. It is i n v o l v e d in maintaining the stability of cell walls and m e m b r a n e s (Marschner 1995). Calcium is an i m p o r t a n t factor in plant signalling as an intracellular second m e s s e n g e r (Bush 1995, W e b b et al. 1996), plant growth and developm e n t (Hepler and W a y n e 1985), as well as metabolism including photosynthesis (Kauss 1987). The photosynthetic activity o f chloroplasts is related to the presence o f Ca 2÷. This e l e m e n t is an important part of the o x y g e n evolving c o m p l e x (Yocum 1991 and refs therein; Bricker and Ghanotakis 1996 and refs therein), as well as a stabilizer of L H C I I apoproteins (Tanaka et al. 1995). T a k i n g into account the indispensability and importance o f Ca 2+ in photosynthesis, we h a v e tried to explain its role in the m e c h a n i s m of Cd 2+ action,

171

E. SKORZY19SKA-POLIT & T. BASZYIVSKI

because these elements are considered to be biological analogues. Although the toxic effects of Cd 2+ on plants are well documented (Barcel6 and Poschenrierder 1990 and refs therein; Wo2ny et al. 1990 and refs therein; Krupa and Baszyriski 1995 and refs therein; Prasad 1996 and refs therein) the mechanism of its cellular action is still unclear. The primary target of Cd 2+ action on the light or dark phase of photosynthesis is the main question being addressed by research groups interested in this subject. In this context, a direct and indirect mechanism of Cd 2+ action on plants was discussed (Krupa and Baszyfiski 1995). The starting point of our studies on the mechanism of Cd 2+ toxic action on the photosynthetic apparatus was the assumption that this metal as a Ca 2+ analogue can remove and/or replace Ca 2+ causing disturbances in growth, development and metabolism of cells. Our p r e s e n t s t u d i e s c o n c e r n the p o s s i b i l i t y o f Cd2+/Ca2+ interaction and its influence on photochemical activity of runner bean plants. W h e n photosynthetic electron transport takes place a decrease of the cytosolic Ca 2+ level is observed and this change may be involved in regulating the activity of key metabolic enzymes including stromal enzymes (see Evans et al. 1991). It is believed that at least two chloroplast enzymes are regulated by stromal Ca 2+ concentration (Muto and Miyachi 1986, Kriemer et al. 1988). In chloroplasts the total (free and complexed) calcium concentration has been determined to be 4 - 23 mM, while stromal free Ca 2+ is maintained at 2.4 - 6.3 pM (Evans et al. 1991 and refs therein). Calcium is taken up into chloroplasts via specific channels. Only one of the three types of the gating mechanism (i. e. control of channel opening and closing) was reported in the thylakoid membranes of Spinacea oleraeea L. (Pottosion and Sch6nknecht 1996). This type, called a voltage-gated channel, increases its open state probability as the membrane potential (the stroma relative to the lumen) becomes positive (see Pifieros and Tester 1997). Calcium uptake by chloroplasts is induced by light (Kreimer et aI. 1985b), changes of Ca 2+ in chloroplasts are very fast (tl/2 = 1 rain) and the maximal yield of this process is 8.3 mmol/L of Ca 2+ taken up per gram of chloroplasts per second (Moore and Ackerman 1984). To measure the fluorescence pa-

172

rameters, dark-adaptation of leaves is necessary to open all energy traps. After that, illumination of leaves causes chlorophyll excitation and its fluorescence. The fast phase of the fluorescence induction curve is related to primary processes of PSII and occurs during the first second of illumination. The slow phase, lasting approximately 5 min, is mainly related to interactions between processes in the thytakoid membranes and metabolic processes in the stroma (Bolh~ir-Nordenkampf and 0quist 1993). Thus, Cd 2+ application at this time may be used to elucidate the mechanism of Cd 2+ penetration into chloroplasts. It seemed to us that Cd 2+ could use Ca 2+ channels for this purpose. W e are also interested in whether modulators of Ca channel activity could cause an increase of Cd transport into chloroplasts because it is documented that Ca 2+ uptake into chloroplasts is stimulated by the ionophore A23187 (Kreimer et al. 1985a).

Material a n d M e t h o d s Plant material and growth conditions Seeds of runner bean plants (Phaseolus coccineus L., cv. Pi~kny Jag) were germinated on wet filter paper in a thermostated darkened chamber (23 °C, 95 % relative humidity). From day 5 the seedlings were cultivated hydroponically (five plants per pot) in aerated Knop nutrient solution. The nutrient solution was composed of (g.m-3): Ca(NO3)2.4H20 1000; KH2PO 4 -280; MgSO4.7H20 - 250; KC1 120; H3BO 3 - 0.2; MnSO 4 - 0.5; CuSO 4 - 0.05; ZnSO 4 - 0.1; (NH4)6MoTO24.4H20 - 0.025. Iron was added as ferric citrate - 25 g.m -3. The plants were grown at 21 °C and PPFD of 120 ~mol m-2.s -1 under a 16-h photoperiod. On day 12 (young plants) and day 22 (older plants) of plant growth in the nutrient solution, the primary leaves were harvested and infiltrated. Infiltration o f p r i m a r y leaves

Discs from primary leaves of plants used to study the toxic effects of Cd 2+ were vacuum infiltrated and then left in infiltration media for 10 min. These media contained different concentrations of CdSO 4 (10 -6, 10 -3, 10 -1 mol.m-3), ionophore A 21387

DOES

(10 -3 mol.m -3) and/or Ca 2+ as CaC12 (10 -t mol.m-3). Deionized water was used as control.

Measurements of ehlorophyll fluoreseenee Modulated Chl a fluorescence was measured at 20 °C using a PAM 101 Chlorophyll Fluorometer (H. Walz, Effeltrich, Germany) equipped with PAM 103 trigger control unit and Schott lamps KL 1500 (FL 101 and FL 103). Prior to measurements, the leaf discs were dark-acclimated at room temperature (20 °C) for at least 30 rain, and air was blown over the leaf surface to minimize the build up of a boundary layer of CO2. The minimal Chl a fluorescence level (Fo) was sensitized by a modulated (1.6 kHz) photon fluence rate of 10 ~tm,m-2.s -1. F' o (minimal fluorescence in steady-state light) was determined in the presence of far-red background light (Balzers 710, Geidenheim/Rhein, Germany) immediately after turning offthe photosynthetic light. The F m and F' m values were obtained by imposing 800 ms Schott lamp flashes. Saturating light pulses of 8400 ~mol.m-2.s 1 (as measured at the leaf position) with 30 s intervals were applied, allowing the fluorescence to relax completely down to the steady-state fluorescence level (Fs) between each flash. Photochemical (qp) and non-photochemical (qN) quenching was calculated according to van Kooten and Snel (1990) and L N U - according to Cornic et al. (1994).

Statistical analysis All experimental data values are means from three independent series, each done with 3 replicates. The data were analysed statistically by one-way analysis of variance (ANOVA) and the Student Newman - Keuls multiple comparisons test for significant difference.

Results

Response of young plants Changes of photochemical activity of young plants infiltrated by media at different concentrations of Cd 2+ (10 -6, 10 -3, 10 -1 mol.m-3), ionophore A23187 (10 -3 mol-m -3) and Ca 2+ (10 -1 mol.m -3) compared

C D 2+ U S E C A 2+ C H A N N E L S

...

with control leaves infiltrated with deionized water with ionophore and Ca ~-+ (at the same concentrations) are presented in Fig. 1 (I, II, III). Concentrations of Cd 2+ of 10 -6 and 10-3mol.m -3 did not change Fv/Fm or Fv/F 0 ratios but 10 -3 mol-m -3 Cd 2+ increased qp to 116 %, decreased L N U to 84 % and qN to 94 % in comparison with the control (column I). The highest Cd 2+ concentration (10-tmol.m -3) reduced photochemical activity of young leaves, decreasing Fv/Fo, Fv/Fm, qp, qN to 83 %, 92 %, 8 1 % , 92 %, respectively. W h e n the control plants (in columns I, II, III) were compared the ionophore was found to increase the efficiency of the photosynthetic apparatus. Infiltration both by ionophore and the lowest Cd 2+ concentration increased qp to 120 %, and decreased L N U to 87 % and slightly decreased Fv/F 0 and qN in comparison with control infiltrated by ionophore (column II). In plants infiltrated by ionophore and 10 -3 mol-m -3 of Cd 2+, Fv/F 0 and L N U decreased to 87 % and 92 %, respectively, and an increase of qp by 25 % in comparison with control. At the highest Cd 2+ concentration in the presence of ionophore, the Fv/Fm and Fv/F0 values decreased to 95 % of that in control. T h e toxic effect of Cd 2+ on Fv/F m and Fv/F 0 was independent on its concentration when the ionophore and Ca 2+ were applied to the medium (column III). The values of qp, qN and L N U were higher in Cdtreated plants in comparison to their control, except L N U value at 10 -3 mol-m -3 Cd 2+, which was the same as control. There were no changes in Fv/Fo and Fv/F m after infiltration of leaves by Ca 2+ (10 -1 mol.m -3) in comparison with conU'ol infiltrated by deionized water, although an increase of qp, qN and L N U to 114 %, 106 % and 109 %, respectively, was observed (Table).

Response of older plants

Leaves of older plants compared to younger, responded with a more marked decrease of Fv/F 0 and Fv/F m values at lower Cd 2+ concentration (Fig. 2: I,. II, III).

173

E. SKORZYI~SKA-POLIT & T. BASZYI~SKI

At 10 -6 mol.m -3 Cd 2+ the Fv/F 0 ratio decreased to 78 % and Fv/F m to 95 % (column I). At 10 -3 mol.m3 Cd2+ these values were 65 % and 90 % in comparison with the control, whilst at 10 -1 mol.m -3 Cd 2+ they decreased to 87 % and 97 %, respectively. With increasing Cd 2+ concentration in the infiltration medium, decreasing qp values were observed (to 80 % for the higher Cd 2+ concentrations as compared to the control). At 10 -6 and 10 -3 mol-m -3 Cd 2+, a decrease o f q N was observed to 92 % and 86 %, respectively, whereas at 10 -l mol.m -3 Cd 2+ it increased slightly in comparison with the control. L N U values increased in Cd 2+ infiltrated plants reaching 115 % of the control at 10 -1 mol.m -3. I

W h e n leaves were infiltrated by ionophore with 10 -3 mol.m -3 Cd 2+ the values o f the fluorescence parameters were approximately the same as in control, with only slight changes o f qp, qN and L N U (column II). The greatest effect on the fluorescence parameters was observed for the lowest and highest Cd 2+ concentration. The value of qp increased to 150 % at 10 -6 mol.m -3 and to 188 % at 10 -1 mol.m -3 Cd 2+, but L N U decreased to 88 % and 76 %, respectively. A decrease o f Fv/F 0 to 73 % and Fv/Fm to 92 % was observed in comparison with the control.

II

+ Cd

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m

Cd, A 23187

+ Cd, A 23187, Ca 5.0 4.5 4.0 3.5 3.0 2.5

0.85 0.80

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10"1

Cd concentration(tool m3)

0

10~

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10-1

A decrease

of qp, Fv/F 0 and qN to 82 %, 96 % and 97 %, respectively, was observed when leaves were infiltrated by Ca 2+ (10 -1 mol.m -3)

0.5

0.5

0.3

174

IIIi

0.6

A p p l i c a t i o n o f additional Ca 2+ to the infiltration medium caused changes dependent on i n c r e a s i n g Cd 2+ concentration (column III). At 10 -1 mol.m -3 Cd 2+, a decrease of Fv/F 0 to 69 % and Fv/F m to 9 1 % was recorded in comparison with the control. A cadmium concentration-dependent increase o f qp and decrease o f L N U were measured, t h e i r v a l u e s at 10 -1 m o l . m -3 Cd 2+ being 187 % and 75 % in relation to the control•

0.3

Fig. 1. Changes in fluorescence parameters in leaves of young runner bean plants infiltrated by • • different Cd2 + concentrations (10-6, 10-3, l0 -I tool.m-3),ionophore A 23187 (10-3 mol.m-3) and calcium (101 mol.m-3) in comparison with the control. Values followed by the same letter are not significantly different at P = 0.05.

D O E S C D 2+ U S E CA 2+ C H A N N E L S ...

in comparison with control plants infiltrated by deionized water (Table). Discussion

Response o f young plants T h e possible existence o f Ca2+/Cd 2+ interactions is

based on studies on lower organisms, where it was f o u n d that the toxic action o f C d 2+ can be limited by

Ca 2+ (Pawlik and Skowrofiski 1994; FernandezPiEas e t a l . 1995). A l s o higher plants s h o w e d changes in C a 2+ c o n t e n t o n treating t h e m with C d 2+ (Greger and L i n d b e r g 1987) and c h a n g e s o f Ca 2+

In Ca2+-free medium, a Ca 2+ channel is characterised by a small selectivity. However, in the presence of Ca 2+, this channel shows a high selectivity for Ca 2+ irrespective of the presence of other ions in the medium (see Tretyn 1994). Therefore, during infiltration Of young leaves by Cd 2+ in the absence of ionophore and Ca 2+, cadmium may reach cytoplasm and/or chloroplasts via some Ca 2+ channels. However, low Cd 2+ concentrations may not cause changes in photochemical activities; moreover, it seems that in their presence electron transport is enhanced (qp increase), which resulted in a decreased fraction of absorbed light not utilized in primary

content in the medium resulted in decreasing or increasing Cd 2+ toxicity (Sk6rzyriska-Polit e t a l .

I +

1998). Antosiewicz et al. (1996) found that in the presence of Cd 2+

II

Cd

+ Cd,

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A 23187

+ Cd, A 23187, C a 5.0

5.0 4.5.

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i

there occurred g e n e expression for Ca 2+ trans-

4"0•

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3.5"

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porting protein to the cell and that uptake of Cd 2+ i n c r e a s e d at a

3.0 •

0.85 0,80 0,75~.~ 0.700.65" 0,60. 0•55.

Before reaching chloroplasts, Cd ions come across plasmatic membranes through channels

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mechanism.

voltage-gated channels were reported in the plasma membrane

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o f different types o f the Stretch-activated

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higher Ca 2+ concentration.

gating

a

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f

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(Pifieros and Tester1997). I~

Fig. 2. Changes in fluorescence parameters in leaves of older runner bean plants infiltrated by different Cd concentrations (10 6 -3 -I -3 • ,10 , 10 mol.m -),mnophore A 23187 (10 -3 mol.m "3) and calcium (10-~ mol.m -3) in comparison with the control. Values followed by the same letter are not significantly different at P = 0.05.

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10-1

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Cd concentration (tool m3)

175

E. SKORZY1VSKA-POLIT & T. BASZYI@KI

Table. Changes in fluorescence parameters in leaves of young and older runner bean plants infiltrated by calcium (101 mol. m-3). The percentage of control is given in parenthesis The values are means (_+SE) from 3 independent series, each in 3 replicates.

Fv/F0

Fv/Fm

qp

qN

LNU

0.802_+0.002(100)

0.693_+0.020(114)

0.574_+0.009(106)

0.551_+0.004(109)

0.804_+0.010 (100)

0.405_+0.060( 8 2 )

0.629_+0.002( 9 7 )

0.694_+0.004(114)

Young plants 4.13_+0.04 ( 1 0 0 ) Older plants 4.07_+0.20 ( 9 6 )

Control leaves were infiltrated by deionized water; their values of fluorescence parameters are in Figures 1 I and 2 I. photochemistry (LNU decrease). Transport of Ca 2+ into the chloroplasts occurs upon illumination via a Ca 2+ uniport transporter associated with the photosynthetic electron transport (Kreimer et al. 1985b, Evans et al. 1991). Calcium ion uptake by chloroplasts takes place only in a medium containing endo- or exogenous ATP (see Tretyn 1994). When ATP utilization is occurring, a more effective photosynthetic transport of electrons is expressed as an increase of qp (Seaton and Walker 1992). We observed that subjecting young leaves to the infiltration medium containing 10-1mol.m -3 Ca 2+ also caused an increase of qp by 14 % (Table). Ions transport into the chloroplast may be linked to consumption of photosynthetically-produced ATE If we assume that Cd 2+ transport occurs via Ca 2+ channels, the characteristics of its uptake would be expected to be similar to that of Ca 2+. This may be indicated by the increase in qp at lower Cd 2+ concentrations. Reduction of the photosynthetic activity of chloroplasts was induced only by the highest Cd 2+ concentration. Changes on the PSII donor side (Fv/F 0 decrease) as well as disturbances in electron transport (decrease of Fv/F m and qp) and increased dissipation of absorbed energy as heat (increase of LNU) took place here at the highest Cd 2+ concentration. In young plants subjected to infiltration with the highest Cd 2+ concentration, the site most sensitive to its action is the PSII donor side. This confirms our earlier studies in vivo, where decreased activity of oxygen evolution (Skdrzyfiska et al. 1991) and release of OEC proteins in Cdtreated plants were observed (Sk6rzyfiska and Baszyfiski 1993).

176

O n additionally applying ionophore during Cd 2+ infiltration, we expected increased metal transport to chloroplasts causing a decrease of photosynthetic activity. This was confirmed by decrease of Fv/F 0 and Fv/F m in plants infiltrated by ionophore and Cd 2+ in comparison to their control (see Fig. 1 II). However, an increase ofqp suggested a more effective electron transport related to ATP consumption, as mentioned earlier, the more so as no changes in qN values were found. The highest Cd 2÷ concentration in the presence of ionophore did not exert such a strong effect on the PSII fluorescence parameters as we expected. This surprising result may be caused by damaging and/or blocking the channels by Cd 2+ ions. Additional application of Ca 2+ in the infiltration medium abolished the effect of Cd 2+ used. Response o f older plants

In mature or in senescencing leaves a high proportion of the total Ca 2+ of the leaf is located in the cell walls (apoplast). Particularly high Ca 2+ concentrations are found in the middle lamella of the cell wall, at the exterior surface of the plasma membrane, in the endoplasmic reticulum and in the vacuole (Marschner 1995). Our earlier studies showed in older plants, a two times higher Ca 2÷ content in primary leaves compared to younger ones (Skdrzyfiska-Polit et al. 1998). At such Ca 2÷ saturation of leaf cells and a low concentration of Cd 2÷ in the infiltrated medium it could be cotransported together with Ca 2+ into the cell interior. However, the highest Cd 2÷ concentration used during infiltration was likely to have caused Ca 2÷ replacement by Cd 2+ at its binding sites. Because Ca 2+ is replace by other cations from its binding

DOES

sites at the exterior surface of the plasma membranes, the calcium requirement increases with increasing external concentrations of heavy metals (see Marschner 1995). The presence of Cd 2+ can effect Ca-dependent changes, ATP formation and electron transport, which is consistent with the qp and Fv/Fm decrease and qN increase fSeaton and Walker 1992). Replacement of Ca 2+ by Cd 2+ in the PSII complex can occur (Matysik etal. 1998), causing changes on its donor side which leads to a decrease of Fv/Fo. Infiltration of leaves of older plants by medium containing only Ca 2+ (10 -1 mol.m -3) also caused decreased photochemical activities of leaves in our experiments (Table). Thus, it can be supposed that the changes in the fluorescence parameters measured already under the influence of higher Cd 2+ concentrations, in older bean plants were connected with those of the Ca 2+ level induced by Cd 2+. It is possible that Ca 2+ was removed by Cd 2+ from its binding sites, and the increased pool of free Ca 2+ caused an increase in the selectivity of the Ca 2+ channels. This would result in a smaller amount of Cd 2+ being transported into the cytosol. In this way a smaller Cd 2+ pool can penetrate into the chloroplasts and Ca 2+ becomes an agent protecting the photosynthetic apparatus from Cd 2+ toxic action. In the presence of ionophore, 10-3 mol.m -3 Cd 2+ does not affect the photochemical activity of leaves of older bean plants. The efficiency of the donor side (decrease of Fv/Fo) and transfer of electrons by PSII (decrease of Fv/Fm) were diminished when the plants were infiltrated by the highest C d 2+ c o n c e n tration in the presence of ionophore. Maybe the increase of qp observed at 10 -1 mol.m -3 Cd 2+ should be associated with effective consumption of ATP used for Ca 2+ transport through Ca 2+ channels outside chloroplasts not to allow its accumulation in chloroplasts. In this case we cannot explain the Fv/F0 decrease following infiltration at 10 -6 mol.m3 Cd2+ in the presence of ionophore. A n addition of Ca 2+ to the infiltration medium with ionophore causes abolition of Cd 2+ toxic action at its lower concentrations on the activity of the PSII donor side (Fv/F0 ratio), whereas at the highest concentration this effect is intensified.

C D 2+ U S E C A 2+ C H A N N E L S

...

A t a higher than optimal Ca 2+ concentration in chloroplasts inhibition of photosynthetic activity is likely to occur. Comparing control plants infiltrated by ionophore with or without Ca 2+ (see Fig. 2 II and III) and infiltrated only by Ca 2+ (Table), we found a reduced efficiency of the photosynthetic apparatus when Ca 2+ was present in the medium. O u r studies concerning the photochemical activity of bean plant chloroplasts, in which leaves were infiltrated by Cd 2+ and ionophore, show that this metal can utilize Ca 2+ channels to penetrate into chloroplasts. The toxic action of Cd 2+ seems to be dependent on the Ca 2+ content in plant cells (what is related with age of plants). Calcium saturation of the binding sites in the apoplast or at the external surface of the cell membrane may determine the effects of C d 2+ action. Depending on its concentration Cd2+can: (i)undergo retention at sites where groups capable of binding divalent cations are present, (ii) pass through cell membranes, utilizing membrane transporters, (iii) induce indirectly a decreased activity of the PSII donor side.

Acknowledgements This paper is a part of a project supportedby the State Committee for ScientificResearch (KBN) grant No. 6P04C.037.09.

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Received May 27, 1999; accepted February 04, 2000

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