Role of Ions in the Regulation of Light-Harvesting

Abstract

Regulation of photosynthetic light harvesting in the thylakoids is one of the major key factors affecting the efficiency of photosynthesis. Thylakoid membrane is negatively charged and influences both the structure and the function of the primarily photosynthetic reactions through its electrical double layer (EDL). Further, there is a heterogeneous organization of soluble ions (K⁺, Mg²⁺, Cl^-) attached to the thylakoid membrane that, together with fixed charges (negatively charged amino acids, lipids), provides an electrical field. The EDL is affected by the valence of the ions and interferes with the regulation of "state transitions," protein interactions, and excitation energy "spillover" from Photosystem II to Photosystem I. These effects are reflected in changes in the intensity of chlorophyll a fluorescence, which is also a measure of photoprotective non-photochemical quenching (NPQ) of the excited state of chlorophyll a. A triggering of NPQ proceeds via lumen acidification that is coupled to the export of positive counter-ions (Mg²⁺, K⁺) to the stroma or/and negative ions (e.g., Cl^-) into the lumen. The effect of protons and anions in the lumen and of the cations (Mg²⁺, K⁺) in the stroma are, thus, functionally tightly interconnected. In this review, we discuss the consequences of the model of EDL, proposed by Barber (1980b) Biochim Biophys Acta 594:253-308) in light of light-harvesting regulation. Further, we explain differences between electrostatic screening and neutralization, and we emphasize the opposite effect of monovalent (K⁺) and divalent (Mg²⁺) ions on light-harvesting and on "screening" of the negative charges on the thylakoid membrane; this effect needs to be incorporated in all future models of photosynthetic regulation by ion channels and transporters.

Keywords: ions; light-harvesting protein complexes; non-photochemical quenching; photoprotection; photosynthesis; state transitions.

Figure 1

A simplified scheme of photophosphorylation in photosynthesis: role of proton and ion heterogeneity. ATP synthase uses proton gradient across the thylakoid membrane; the proton transport across the thylakoid is coupled to light-driven electron transport (dotted black lines). The proton gradient forms electrochemical gradient of protons [pmf —proton motive force, which includes Δψ (the membrane potential component) and ΔpH (proton gradient component; see Equation 1)]; pmf is the driving force for ATP synthesis. The most accepted view of photophosphorylation is: “delocalized phosphorylation”—equilibrated concentration of protons in the bulk (in the stroma/lumen) is used (original Mitchell theory—dashed blue lines, see Mitchell, 1966). A second less accepted view is that of “localized phosphorylation” (dashed red lines, see Mulkidjanian et al., 2006): protons from the local domains in lumen/stroma that are in the close vicinity of membrane (see Area I) are involved in ATP synthesis (Dilley, 2004). Localized protons interact with ions attached to fixed membrane charges, mostly Mg²⁺ (note: K⁺ and Cl⁻ are more abundant in the thylakoid stroma/lumen bulk, see Barber, 1980b). The distribution of the dominant ions (K⁺, Mg²⁺, Cl⁻) in the local (Area I) and bulk domain (Area II) areas is controlled by properties of the electrical double layer—EDL (see Cevc, 1990); EDL is characterized by the ion profile around the membrane, as well as the electrical field around the membrane, between ψ_o and ψ_bulk. (cf. Figure 2). Photosynthetic proton pumping into the lumen is accompanied by counter-ion transport from lumen into stroma. The process of electron transport and the use of protons by ATPase can be uncoupled by the addition of various ionophores (nigericin—electroneutral antiporter H⁺/K⁺; valinomycin—K⁺ ionophore; A23187—Mg²⁺) that can disrupt the membrane potential and ion/proton gradients. The efficient activity of the particular ionophore in uncoupling requires the presence of appropriate cationic species at the membrane surface (Barber, 1980b); therefore, their uncoupling ability differs between high & low screening modes when more Mg²⁺ and K⁺ are attached to thylakoid membranes (see Figure 5).

Figure 2

A schematic representation of charge/electrostatic field distribution around thylakoid membranes that forms electric double layer (EDL). Negatively charged amino acid residues form fixed charges on the thylakoid membrane surface that is screened by positive ions (mostly Mg ²⁺) in the lumen as well as in the stroma. The concentration of screening cations decreases with distance from the membrane surface in contrast to that of the anions that are more abundant farther from the negative charges of the membrane. This charge distribution then forms the electric double bilayer (EDL) where the electrical field (see blue line) on the luminal/stromal surface of thylakoid membrane (ψ_o-lumen, ψ_o-stroma) is higher than the electrical field measured in the bulk (ψ_bulk^lumen; ψ_bulk^stroma); there is also a characteristic distribution (see dotted magenta lines) in the concentration of cations at the membrane [C⁺]_o and in the bulk [C⁺]_bulk. EDL then represents asymmetric charge distribution of both the anions (e.g., Cl⁻) and the cations (K⁺; Mg²⁺) caused by fixed charges on the thylakoid membrane; the concentration of the anions/cations progressively increases/decreases with the distance from the charged thylakoid membrane surface (Barber et al., 1977). The difference between ψ_bulk-ψ_o is characterized by the extent of electrostatic screening—the higher the electrostatic screening by the ions the lower is the observed difference.

Figure 3

Chlorophyll a fluorescence measurements in photosynthetic organisms. (A) (top): A protocol used for the detection of PSII photochemistry upon exposure with ~500 μmol photons m⁻²s⁻¹ light (Krause and Weis, 1991). The minimal level of Chl a fluorescence of open PSII reaction centers plus that from PSI is F_o; it is measured in a dark adapted sample at very low (“measuring”) light (~5 μmol photons m⁻²s⁻¹). The maximal fluorescence in dark-adapted sample is F_m, but its changed value during actinic light is referred to as F_m′, and after the actinic light period light, it is labeled as F_m″. In the experiment shown, it was measured with high intensity multiple turnover (MT) flashes (~1500 μmol photons m⁻²s⁻¹, flash duration 200 ms), given for a short period (~200 ms). (Note: MT flash induces multiple events of charge separation in PSII.) The fluorescence increase during the MT flash has a characteristic polyphasic rise to a plateau or a peak (see inserts in both panels A and B). The F_m and F_m′ (as well as F_m″) values are used for the calculation of PSII photochemistry as well as for non-photochemical quenching (NPQ) of the excited state of Chl a, the latter equals (Fm-Fm′)/Fm. Black bars (near the abscissa) represent periods without actinic irradiation (i.e., darkness), whereas during the open (clear) bar, actinic light is on. The sample used in the experiment shown here was Rhodomnas salina cells (from Kaňa et al., 2012b). (B) Chl a fluorescence transient measured at high intensity (~5000 μmol photons m⁻²s⁻¹) MT flash (from Kaňa et al., 2008). The inset shows Chl a fluorescence transient in a short, 100 μs long, single turnover (ST) flash, at very high irradiation (~100 000 μmol photons m⁻²s⁻¹). Using the MT flash at high light intensity, we observe a polyphasic O-J-I-P fluorescence transient, where the O–J rise is due to primary photochemical reactions, the subsequent J–I–P transient being the thermal phase (cf. Stirbet and Govindjee, 2012). The fluorescence rise in single turnover (ST) flash (see inset in A) is the fast O-J-I-P fluorescence change during the single charge separation event induced by the ST flash that closes all the PSII reaction centers in a very short period (in about 30–100 μs) due to the extremely high intensity of light (~100,000 μmol photons m⁻²s⁻¹, duration 100 μs). We note that explanation of experimental differences between ST and MT fluorescence parameters, obtained with single and multiple turnover flashes, requires detailed knowledge of the studied model organism (see e.g., Kolber et al., ; Koblížek et al., 2001).

Figure 4

Ion induced changes in variable chlorophyll a fluorescence in isolated thylakoids (data adapted from Vandermeulen and Govindjee, 1974). Time course of cation induced changes in chlorophyll a fluorescence in oat chloroplast suspension containing 5 μg chlorophyll ml⁻¹, and 5 μM DCMU in low salt buffer. Subsequently, 20 mM NaCl and 1 mM MgCl₂ were added (see arrows).

Figure 5

A simplified scheme of changes in protein organization in different states of electrostatic screening (adapted from Barber, 1980b). The table summarizes basic conditions for low or high screening—high screening is induced by high divalent cation content [e.g., >220 μM Mg²⁺ with no (K⁺), >440 μM Mg²⁺ with 10 mM (K⁺), see (Mills and Barber, 1978) for details]. The switch between low/high screening mode is a complex interplay between monovalent/divalent cation concentrations as it is described by EDL model of thylakoid membrane (Barber et al., 1977). The low/high screening state is then reflected in various physiological processes including intensity of Chl a fluorescence (low intensity /high intensity), thylakoid membrane stacking or separation, lower/high excitation spillover between photosystems, State II/State I, protein repulsion/aggregation, proposed role in non-photochemical quenching, preferable uncouplers (Mg²⁺ ionophore A23187 vs. K⁺/H⁺ antiporter nigericin), effect on electron-proton coupling in photophosphorylation, effect on PSII photochemistry. The screening is caused by ion attachment to membranes that results in screening of the electric-field of the membrane charge (i.e., damping of electrostatic field of fixed charges caused by the presence of interacting ions). (A). Simplified scheme of membrane protein organization in the state of low electrostatic screening. Green cycles represent negatively charged proteins (PSI complex, light harvesting antennas), cyan particles represent less charged PSII. Monovalent cation K⁺ is shown as a dominant ion attached to the negatively charged membrane surface. (B) A simplified scheme of membrane protein organization in the state of high electrostatic screening, divalent cation, Mg²⁺, is proposed to be the main ion attached to the negatively charged membrane surface. Role of protein phosphorylation on protein redistribution is indicated (^# values were taken from Mills and Barber, 1978).