The high-energy state of the thylakoid system as indicated by chlorophyll fluorescence and chloroplast shrinkage

Certain long-term fluorescence phenomena observed in intact leaves of higher plants and in isolated chloroplasts show a reverse relationship to light-induced absorbance changes at 535 nm (“chloroplast shrinkage”).

1. 1. In isolated chloroplasts with intact envelopes strong fluorescence quenching upon prolonged illumination with red light is accompanied by an absorbance increase. Both effects are reversed by uncoupling with cyclohexylammonium chloride.

2. 2. The fluorescence quenching is reversed in the dark with kinetics very similar to those of the dark decay of chloroplast shrinkage.

3. 3. In intact leaves under strong illumination with red light in CO₂-free air a low level of variable fluorescence and a strong shrinkage response are observed. Carbon dioxide was found to increase fluorescence and to inhibit shrinkage.

4. 4. Under nitrogen, CO₂ caused fluorescence quenching and shrinkage increase at low concentrations. At higher CO₂ levels fluorescence was increased and shrinkage decreased.

5. 5. In the presence of CO₂, the steady-state yield of fluorescence was lower under nitrogen than under air, whereas chloroplast shrinkage was stimulated in nitrogen and suppressed in air.

6. 6. These results demonstrate that the fluorescence yield does not only depend on the redox state of the quencher Q, but to a large degree also on the high-energy state of the thylakoid system associated with photophosphorylation.

A one microsecond component of chlorophyll luminescence suggesting a primary acceptor of system II of photosynthesis different from Q

The kinetics of the luminescence of chlorophyll a in Chlorella vulgaris were studied in the time range from 0.2 μs to 20 μs after a short saturating flash ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 25}\text{ns}$) under various pretreatment including anaerobiosis, flashes, continuous illumination and various additions. A 1 μs luminescence component probably originating from System II was found of which the relative amplitude was maximum under anaerobic conditions for reaction centers in the state SPQ^? before the flash, about one third for centers in the state S⁺PQ^? or SPQ before the flash, and about one tenth for centers in the state S⁺PQ before the flash. S is the secondary donor complex with zero charge; S⁺ is the secondary donor complex with 1 to 3 positive charges; P, the primary donor, is the photoactive chlorophyll a, P-680, of reaction center 2; Q^? is the reduced acceptor of System II, Q. Under aerobic conditions, where an endogenous quencher presumably was active, the luminescence was reduced by a factor two.The 1 μs decay of the luminescence is probably caused by the disappearance of P⁺ formed in the laser flash according to the reaction ZP⁺ → Z⁺P in which Z is the molecule which donates an electron to P⁺ and which is part of S. After addition of hydroxylamine, the 1 μs luminescence component changed with the incubation time exponentially (τ = 27 s) into a 30 μs component; during the same time, the variable fluorescence yield, measured 9 μs after the laser flash, decreased by a factor 2 with the same time constant. Hereafter in a second much slower phase the fluorescence yield decreased as an exponential function of the incubation time to about the dark value; meanwhile the 30 μs luminescence increased about 50% with the same time constant (τ = 7 min). Heat treatment abolished both luminescence components.The 1 μs luminescence component saturated at about the same energy as the System II fluorescence yield 60 μs after the laser flash and as the slower luminescence components. From the observation that the amplitude is maximum if the laser flash is given when the fluorescence yield is high after prolonged anaerobic conditions (state SQ^?), we conclude that the 1 μs luminescence is probably caused by the reaction

$\text{PWQ}{}^{\mathrm{?}}\text{+hv →}\text{P}\text{1}\text{WQ}{}^{\mathrm{?}}\text{→}\text{P}{}^{\mathrm{+}}\text{W}{}^{\mathrm{?}}\text{Q}{}^{\mathrm{?}}\text{→}\text{P}\text{1}\text{WQ}{}^{\mathrm{?}}\text{→ PWQ}{}^{\mathrm{?}}\text{+hv}$

in which W is an acceptor different from Q. The presence of S⁺ reduced the luminescence amplitude to about one third. Two models are discussed, one with W as an intermediate between P and Q and another, which gives the best interpretation, with W on a side path.     

Iodination of chloroplasts. I. Properties of iodinated chloroplasts

Spinach chloroplasts exposed to iodide can be washed free of the bulk of the iodide. In the presence of lactoperoxidase and H₂O₂, iodide can be introduced into chloroplasts in high amounts and in non diffusible forms. The resultant particles, which have been named iodochloroplasts, extrude their iodide upon stimulation by light. The form and the amount of extruded iodide bears a definite relationship to the amount of incident light. A flash of marginally effective light is additive to the next such flash even after a lapse of 10 min of darkness. These and other properties of iodochloroplasts may make them of great use in the study of intermediate reactions of photosynthesis.     

Characterization of the low temperature thermoluminescence band Zv in leaf an explanation for its variable nature

Out of the six thermoluminescence bands reported for a mature leaf, one band (Z_v) appearing at the lowest temperatures is dependent on the temperature of illumination. The characteristics of this band in fresh leaf are compared with those in a leaf heated to 60°C for 5 min. It is concluded here that this band, following illumination at temperatures lower than 173 K, is part of Arnold and Azzi's Z band (Arnold, W. and Azzi, J.R. (1971) Photochem. Photobiol. 14, 233–240). However, it is a part of peak I when observed subsequent to illumination beyond 173 K. An explanation for the appearance of this band at different temperatures is proposed.     

Induction kinetics of delayed light emission in spinach chloroplasts

Induction curves of the delayed light emission in spinach chloroplasts were studied by measuring the decay kinetics after each flash of light. This study differs from previous measurements of the induction curves where only the intensities at one set time after each flash of light were recorded. From the decay kinetics after each flash of light, the induction curves of the delayed light emission measured 2 ms after a flash of light were separated into two components: one component due to the last flash only and one component due to all previous flashes before the last one. On comparing the delayed light induction curves of the two components with the fluorescence induction curves in chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and in chloroplasts treated with hydroxylamine and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the component due to the last flash only is found to be dependent on the concentration of open reaction centers and the component due to all previous flashes except the last is dependent on the concentration of closed reaction centers. This implies that the yield of the fast decaying component of the delayed light emission is dependent on the concentration of open reaction centers and the yield of the slow decaying component is dependent on the concentration of closed reaction centers.     

Role of β-carotene in the reaction centres of Photosystems I and II of spinach chloroplasts prepared in non-polar solvents

Spinach chloroplasts have been prepared nonaqueously using non-polar solvents (n-hexane, CCl₄, n-heptane) and the β-carotene content extracted in a controlled manner. This procedure is reproducible and does not result in large structural or spectral changes of the chloroplasts. The organisation of the chlorophyll-proteins is unaltered, as fragmentation with digitonin results in the appearance of the same fractions as found previously for aqueously-prepared chloroplasts, including the pink zone containing cytochromes f and b₆ in the ratio 1:2. The chloroplasts possess both Photosystem I activity (P-700 photo-bleaching, and NADP⁺ photoreduction) and Photosystem II activity (parabenzoquinone reduction with Mn²⁺ as electron donor, and chlorophyll fluorescence induction). Use of moderate intensity red illumination has allowed a study of the role of β-carotene in photochemistry separate from its roles in energy transfer and photoprotection.Removal of the fraction of β-carotene closely associated with the Photo-system I reaction centre caused the rate of NADP⁺ photoreduction to fall to a low, but significantly non-zero level. Thus, in the complete absence of β-carotene, photochemistry can still be observed, however the specific association of β-carotene with the reaction centre is required for maximal rates. We propose that β-carotene bound at the reaction centre decreases the rate of transfer of excitation energy away from the reaction centre, and increases the rate of photochemistry. It is possible that this occurs via formation of an exciplex between ground state β-carotene and chlorophyll in the first excited state.     

Increase effected by calcium ion in the rate of oxygen evolution from preparations of Phormidium luridum

The presence of Ca²⁺ causes a twentyfold or greater increase in the rate of oxygen evolution by cell-free preparations of Phormidium luridum. The requirement for Ca²⁺ is specific; other divalent cations are much less effective or are inhibitory. The rate of the Hill reaction is maximal at 30 mM CaCl₂ in both detergent-free and Brij 35 preparations. The 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive component of oxygen-evolving activity in each preparation also shows the requirement for added Ca²⁺. This indicates that Ca²⁺ is acting close to the oxygen-evolving reaction center of Photosystem II. Defatted bovine serum albumin increases the rate of oxygen evolution in the detergent-free preparation, but does not compete with Ca²⁺, discounting fatty acid mediation of the effects of Ca²⁺. Neither excess Hill acceptor nor uncouplers of photophosphorylation diminish the stimulatory effects of Ca²⁺.     

Identification of S2 as the sensitive state to alkaline photoinactivation of Photosystem II in chloroplasts

1. Chloroplasts have been preilluminated by a sequence of n short saturating flashes immediately before alkalinization to pH 9.3, and brought back 2 min later to pH 7.8. The assay of Photosystem II activity through dichlorophenolindophenol photoreduction, oxygen evolution, fluorescence induction, shows that part of the centers is inactivated and that this part depends on the number of preilluminating flashes (maximum inhibition after one flash) in a way which suggests identification of state S₂ as the target for alkaline inactivation.2. As shown by Reimer and Trebst ((1975) Biochem. Physiol. Pflanz. 168, 225–232) the inactivation necessitates the presence of gramicidin, which shows that the sensitive site is on the internal side of the thylakoid membrane.3. The electron flow through inactivated Photosystem II is restored by artificial donor addition (diphenylcarbazide or hydroxylamine); this suggests that the water-splitting enzyme itself is blocked. The inactivation is accompanied by a solubilization of bound Mn²⁺ and by the occurrence of EPR Signal II “fast”.4. Glutaraldehyde fixation before the treatment does not prevent the inactivation which thus does not seem to involve a protein structural change.     

Membrane surface potential and the reactivity of the System II primary electron acceptor to charged electron carriers in the medium

A hypothesis is proposed to explain the change in the apparent rate constant for the reaction between the primary electron acceptor of System II situated in the thylakoid membrane and the artificial electron acceptors added in the medium. Dark oxidation rate of the primary acceptor by artificial electron acceptors was monitored by measuring the induction of chlorophyll fluorescence in the presence of an electron transport inhibitor, 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, in spinach chloroplasts. The apparent rate constant for the oxidation changed widely when the medium pH or salt concentrations were varied, or ionic detergents were added. The change was quantitatively ascribed (1) to the change in the local concentration of electron acceptors at the thylakoid surface due to the electrical potential difference between the surface and the bulk aqueous phase (Gouy-Chapman diffuse double layer theory) and (2) to the situation whereby the apparent rate constant is determined with respect to concentration in the bulk phase.Values for the surface potential in the vicinity of System II were estimated from the change in the apparent rate constant under various conditions. The results closely agreed with those obtained previously from the rate constant of the dark step of the System II-dependent Hill reaction with ferricyanide (Itoh, S. (1978) Plant Cell Physiol. 19, 149–166).Application of the hypothesis to various reactions between the added ionic reagents and the endogenous components in the membrane or between the endogenous components situated in different parts of the membrane is discussed.