ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 1, с. 38-42
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
YM 581.1
THE INTERRELATIONSHIP BETWEEN HCO3 , NO3 AND Cl- AS EFFECTIVE ANIONS IN THE PHOTOSYNTHETIC ELECTRON TRANSPORT1
© 2014 M. E. H. Osman
Botany Department, Faculty of Science, University of Tanta, Tanta, Egypt Received December 2, 2012
Thylakoids were isolated from the leaves of three different plants (Pisum sativum L., Lactuca sativa L., Rapha-nus sativus L.). The addition of HCO3 to a suspension of salt- and HCO3 -depleted thylakoids (suspended in salt-free medium) raised the rate of O2 evolution up to fourfold. This stimulation could be partially replaced by the addition of chloride or nitrate ions. However, the addition of HCO- in the presence of Cl- or NO- resulted in a higher stimulation of O2 evolution (sixfold in the presence of nitrate and sevenfold in the
presence of chloride). On the other hand, the addition of HCO- to the thylakoids depleted from salt only raised the rate of O2 evolution by 10-15%, whereas 40-70% was obtained by the addition of nitrate or chloride ions. The fluorescence induction studies indicated a significant decrease in the yield of the variable fluorescence of the salt- and HCO- -depleted thylakoids. A partial increase in the fluorescence yield was obtained by the addition of HCO- alone. A typical fluorescence induction curves were obtained by the addition
of HC O 3 in the presence of Cl or NO3 ions. The obtained data suggest a similar role for chloride and nitrate ions in O2 evolution in the Hill reaction, which is restricted at the donor side of photosystem II, whereas bicarbonate plays its role at both sides (acceptor and donor sides). The presented data are those obtained for the thylakoids of P. sativum, which were more or less similar to those obtained for L. sativa and R. sativus.
Keywords: Pisum sativum - chloride - nitrate - bicarbonate -photosynthetic electron transport
DOI: 10.7868/S0015330314010096
INTRODUCTION gested that bicarbonate participates in the formation
During the last decades, intensive studies have been of,Mn clus*ers ?pable of water oxidation or serves as a
performed to elucidate the possible role of some an- substrate for the water-^mng crnter. In oher
ions in the O2 evolution in the Hill reaction. The sti- ^^urnrationis, Khm°v et al. [6] and Yrnda et al[7j
_ reported that bicarbonate may be an essential ligand of
mulatory effect of HCO- on the evolved O2 was indi- the functional Mn clusters in the O2-evolving com-cated by many researchers [1]. The site of the action of plex. Allakhverdiev et al. [8] concluded that bicarbon-
this ion in the primary photosynthetic process is still a ate was an essential constituent of the water-oxidizing
controversy. Govindjee et al. [2] reported that the ma- complex of PSII, important for its assembly and main-
jor site of bicarbonate must be located behind the pri- tenance in the functionally active state. mary electron acceptor "Q" of the photosystem II
(PSII), while Metzner [3, 4] suggested a catalytic role A pr°tective rob of M^Aornte for the d°n°r side
- of the PSII against photoinhibition and thermoinacti-
°f HCO3 at the don°r side °f the °2-ev°lving system. vation was reported by Klimov et al. [9]. Pobeguts et al.
In addition, bicarbonate requirement for the donor [10] indicated a protective effect of bicarbonate
side was demonstrated by Klimov et al. [5], who sug- against the extraction of the extrinsic proteins of the
---water-oxidizing complex from PSII membrane frag-
This text was ssuimtaed by the author in English. ments. Furthermore, on the basis of fluorescence
studies, Pobeguts et al. [11] discussed a possible bicarbonate binding site and its physiological role within the water-oxidizing complex of PSII. On the other
Abbreviation', photosystem - PS.
Corresponding author, Mohamed El-Anwar H. Osman. Botany Department, Faculty of Science, University of Tanta, Tanta, Egypt. E-mail: elanwar_osman@yahoo.com hand, Umena et al. [12] showed in their crystallo-
THE INTERRELATIONSHIP BETWEEN HCO3 , NO3 AND Cl- AS EFFECTIVE
39
Table 1. Effect of different anions on the salt- and HC O3 -depleted thylakoids
Treatment O2 evolution, ^mol O2/(mg Chl h) Treatment effect
Control (salt depleted) 7.2 ± 0.9 -
+ 5 mM HC O3 29.2 ± 1.5 4.1
+ 10 mM Cl- 15.3 ± 1.8 2.1
+ 10 mM N O3 11.9 ± 1.1 1.6
+ 5 mM HC O3 + 10 mM Cl- 53.8 ± 1.2 7.5
+ 5 mM HC O3 + 10 mM N O3 43.1 ± 1.8 6.0
+ 5 mM HC O3 + 10 mM N O3 + 10 mM Cl- 60.0 ± 1.7 8.3
Notes: Reaction mixture contained 0.4 M sucrose, 20 mM Hepes-KOH buffer (pH 6.6), 1 mM FeCy, and thylakoids suspension equivalent to 38 ^g Chl/mL. Values represent mean values ± standard deviation (n = 5). The anions were added as potassium salts.
graphic studies that bicarbonate was bound only in the vicinity of the non-heme iron on the acceptor side of PSII. Moreover, Govindjee and Shevela [13] reported that bicarbonate functions both on the electron acceptor as well as on the electron donor side of PSII and does not exert any effect on PSI. The requirement of chloride for the primary photosynthetic process was also well established by Kelley and Izawa [14]. They indicated that the chloride ion was required in the O2 evolution step at the donor site of PSII. Further studies by Wincencjuss et al. [15] showed the participation of chloride in dioxygen-producing transition from S3 to S0 in the O2-evolving system. Recent structural evidence from cyanobacterial PSII indicated that there was at least one chloride-binding site in the vicinity of the oxygen-evolving complex [16]. Also, Umena et al. [12] suggested that at least two binding sites of chloride occurred in the vicinity of the oxygen-evolution system at the donor side of PSII. A catalytic role of nitrate in the donor side of PSII was reported by Osman et al. [17, 18] and Osman and El-Nagar [19].
This work was carried out to test the ability of these different anions to replace each other and to illustrate their interrelationships.
MATERIALS AND METHODS
Thylakoids were isolated from the leaves of three different plant species (Pisum sativum L., Lactuca sativa L., and Raphanussativus L.), as described by Osman [17]. To deplete them from salts (except bicarbonate), they were washed for 1 min in 0.35 M sucrose solution in a 20 mM Hepes-KOH buffer (pH 8.0). To minimize the bicarbonate content they were treated with a medium containing 0.3 M sucrose and 100 mM sodium format in 50 mM MES/ KOH buffer (PH 5.0).
CO2 gas. To remove CO2, the suspension was flushed by stream of nitrogen (5 min above the suspension). Afterwards, it was centrifuged at 3000 g for 5 min. The suspension was carefully resuspended under nitrogen in the same medium containing the tested anions, and the pH of the suspension was adjusted to 6.6. The probe was incubated under N2 in the dark for 5 min before measurement. The chlorophyll concentration of the used thylakoid suspension was estimated according to Metzner et al. [20].
The rate of O2 evolution was measured at 20 °C in the presence of 1 mM FeCy as an electron acceptor with a Clark type electrode, as described by Osman [17]. The light intensity was 300 ^E/(m2 s).
Fluorescence induction measurements were carried out at room temperature (20 ± 1°C) in the absence of FeCy, according to a method described by Franck et al. [21]. The actinic light was supplied by a helium-neon laser (632.8 nm), switched on by magnetic shutter (0.6 ms opening time), and focused on the sample cuvette. The light intensity at the sample level was 0.7 W/m2. Fluorescence emission was filtered through the photomultiplier (Rubilith-RTCXP 1002). Variation of the signal as a function of time were recorded on the memory screen of a Tektronix Oscilloscope (Type Tektronix 546 B). A dark adaptation time of 10 min between measurements was sufficient for complete relaxation of the photosynthetic electron flow of the thylakoid samples between the light periods.
The presented data are those obtained for the thy-lakoids of P. sativum, which were more or less similar to those obtained for L. sativa and R.. sativus.
RESULTS
Table 1 summarizes the effects of the tested anions
By this means the bound HCO3 was converted into on the O2 production ofa suspension, which was com-
Table 2. Effect of different anions on salt-depleted thylakoids
Treatment O2 evolution, ^mol O2/(mg Chl h) Anion effect
Control (salt depleted ) 43.0 ± 1.5 -
+ 10 mM Cl- 71.0 ± 1.0 1.7
+ 10 mM N O- 60.2 ± 2.0 1.4
+ 10 mM HC O- 48.0 ± 2.0 1.1
Notes: Reaction condition as in table 1, except chlorophyll concentration, which was 42 ng Chl/mL. "Vklues represent mean values ± standard deviation (n = 5). The anions were added as potassium salts.
pletely deprived of salts, including HC O3 (measured in a salt-free medium, pH 6.6). The obtained results showed that the addition of HC O- stimulated the O2 evolution by a factor of 4, while a weak stimulation could be observed if bicarbonate was replaced by nitrate or chloride. The addition of bicarbonate in the presence of chloride or nitrate induced the increase in the amount of the evolved oxygen, more than sevenfold in the presence of Cl- and sixfold in the presence of nitrate. The greatest increase in the O2 evolution capacity could be observed if the medium was completed by the addition of all three anions.
Table 2 shows the effect of these anions on salt- but not bicarbonate-depleted thylakoid suspensions. The data showed a low stimulation by further addition of
HC O - , whereas significant stimulation could be observed with nitrate and chloride, 1.7-fold and 1.4-fold in the case of Cl- and NO- , respectively.
Figure 1 shows the fluorescence time course of thylakoid preparations, which were suspended in completely salt-free media after the addition of chloride or nitrate ions. The obtained fluorescence curves show an increase in the yield of the variable fluorescence both after the addition of N O- or Cl-. As can be seen from
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