Excitation-energy distribution in green algae. The existence of two independent light-driven control mechanisms

Three distinct states can be identified for cells of the green alga Chlorella vulgaris; State 1 and State 2 obtained by preillumination in far-red and red light, respectively, and the dark state obtained by dark-adaptation. Addition of the inhibitor DCMU to algal cells leads to an initial rapid increase in chlorophyll-a fluorescence reflecting the closure of Photosystem II traps. This, in the case of dark and state-2-adapted algae is followed by a slow light-dependent increase to a fluorescence yield typical of State-1-adapted cells. Measurements of low temperature (77 K) emission spectra indicate that the low fluorescence yields of dark and State-2-adapted algae reflect similar balances in excitation-energy distribution between the two photosystems. In both cases, the balance favours PS I and the slow fluorescence increase seen in the poisoned algae reflects a redressing of this balance in favour of PS II. The low fluorescence yield of State-2-adapted algae is thought to be associated with the phosphorylation of chlorophyll a/b light-harvesting protein (Biochim. Biophys. Acta (1983) 724, 94–103). Measurements of the uncoupler and ATPase sensitivity of the light-dependent increases seen in DCMU-poisoned cells indicate that the low fluorescence yield of dark-adapted algae is of different origin. Evidence is presented showing that the light-driven changes in excitation-energy distribution seen in green algae involve two distinct processes; a low-intensity, wavelenght-independent change reflecting simple light/dark changes and a higher intensity, wavelength-dependent change reflecting State 1/State 2 adaptation. The former changes appear to be associated with changes in the local ionic environment within the algal chloroplast, whilst the latter appear to reflect changes in the phosphorylation state of chlorophyll a/b light-harvesting protein.     

Orientation of photosystem-I pigments. Investigation by low-temperature linear dichroism and polarized fluorescence emission

Using absorption and fluorescence experiments at low temperature with polarized light on oriented samples, the orientation of PS-I-related pigments, both in green plants and in Chlamydomonas reinhardtii, has been investigated on isolated pigment-protein complexes and intact thylakoids. The following observations have been made. (i) The isolation procedure of PS I₁₁₀, PS I₆₅, LHC I and CP0) particles from pea and C. reinhardtii do not alter significantly the intrinsic orientation of the pigments inside the complexes; (ii) Chl b is a structural component of PS I, linked to the peripheral antenna, with an orientation with respect to the thylakoid plane different from that observed in the main light-harvesting complex (iii) PS I₆₅ (i.e., ‘core’ PS I) of pea and C. reinhardtii contains identical chromophores having the same orientation with respect to the geometrical longest axis (axes) of the complexes. (iv) LHC I and CP0 (i.e., PS I ‘peripheral antenna’) of pea and C. reinhardtii have identical oriented chromophores, except that a long-wavelength component with a high anisotropy is only present in green plants. This set of pigments, which absorbs at 705–725 nm, has the same orientation as the dipoles emitting F₇₃₅ and also as the Q_Y transition of P-700. (v) All the long-wavelength fluorescence properties of the various studied membranes are explained by these data on isolated PS I complexes: wild-type C. reinhardtii and Chl-b-less barely fluoresce from the core pigments, while a CP1 deficient mutant of C. reinhardtii and wild-type barley fluoresce from the antenna pigments.     

EPR detection of a cryogenically photogenerated intermediate in photosynthetic oxygen evolution

In Photosystem II preparations at low temperature we were able to generate and trap an intermediate state between the S₁ and S₂ states of the Kok scheme for photosynthetic oxygen evolution. Illumination of dark-adapted, oxygen-evolving Photosystem II preparations at 140 K produces a 320-G-wide EPR signal centered near g = 4.1 when observed at 10 K. This signal is superimposed on a 5-fold larger and somewhat narrower background signal; hence, it is best observed in difference spectra. Warming of illuminated samples to 190 K in the dark results in the disappearance of the light-induced g = 4.1 feature and the appearance of the multiline EPR signal associated with the S₂ state. Low-temperature illumination of samples prepared in the S₂ state does not produce the g = 4.1 signal. Inhibition of oxygen evolution by incubation of PS II preparations in 0.8 M NaCl buffer or by the addition of 400 μM NH₂OH prevents the formation of the g = 4.1 signal. Samples in which oxygen evolution is inhibited by replacement of Cl^? with F^? exhibit the g = 4.1 signal when illuminated at 140 K, but subsequent warming to 190 K neither depletes the amplitude of this signal nor produces the multiline signal. The broad signal at g = 4.1 is typical for a $\text{S =}\text{5}\text{2}$ spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.     

The chloride requirement for photosynthetic oxygen evolution. Analysis of the effects of chloride and other anions on amine inhibition of the oxygen-evolving complex

In the presence of Cl^?, the severity of ammonia-induced inhibition of photosynthetic oxygen evolution is attenuated in spinach thylakoid membranes (Sandusky, P.O. and Yocum, C.F. (1983) FEBS Lett. 162, 339–343). A further examination of this phenomenon using steady-state kinetic analysis suggests that there are two sites of ammonia attack, only one of which is protected by the presence of Cl^?. In the case of Tris-induced inhibition of oxygen evolution only the Cl^? protected site is evident. In both cases the mechanism of Cl^? protection involves the binding of Cl^? in competition with the inhibitory amine. Anions (Br^? and NO^?₃) known to reactive oxygen evolution in Cl^?-depleted membranes also protect against Tris-induced inhibition, and reactivation of Cl^?-depleted membranes by Cl^? is competitively inhibited by ammonia. Inactivation of the oxygen-evolving complex by NH₂OH is impeded by Cl^?, whereas Cl^? does not affect the inhibition induced by so-called ADRY reagents. We propose that Cl^? functions in the oxygen-evolving complex as a ligand bridging manganese atoms to mediate electron transfer. This model accounts both for the well known Cl^? requirement of oxygen evolution, and for the inhibitory effects of amines on this reaction.     

Measurement of the relative electron-transport capacity of Photosystem I and Photosystem II in spinach chloroplasts

The ratio of Photosystem (PS) II to PS I electron-transport capacity in spinach chloroplasts was compared from reaction-center and steady-state rate measurements. The reaction-center electron-transport capacity was based upon both the relative concentrations of the PS II_α, PS II_β and PS I centers, and the number of chlorophyll molecules associated with each type of center. The reaction-center ratio of total PS II to PS I electron-transport capacity was about 1.8:1. Steady-state electron-transport capacity data were obtained from the rate of light-induced absorbance-change measurements in the presence of ferredoxin-NADP⁺, potassium ferricyanide and 2,5-dimethylbenzoquinone (DMQ). A new method was developed for determining the partition of reduced DMQ between the thylakoid membrane and the surrounding aqueous phase. The ratio of membrane-bound to aqueous DMQH₂ was experimentally determined to be 1.3:1. When used at low concentrations (200 μM), potassium ferricyanide is shown to be strictly a PS I electron acceptor. At concentrations higher than 200 μM, ferricyanide intercepted electrons from the reducing side of PS II as well. The experimental rates of electron flow through PS II and PS I defined a PS II/PS I electron-transport capacity ratio of 1.6:1.     

EPR studies on the photosystem II donor side in salt-washed and reconstituted inside-out thylakoids

EPR measurements on inside-out thylakoids revealed that salt-washing, known to inhibit oxygen evolution and release a 23 and a 16 kDa protein, induced a Signal II_f and decreased the EPR signal from state S₂. Readdition of the released 23 kDa protein restored the oxygen evolution and decreased the Signal II_f, but did not relieve the decrease in the state S₂ signal. It is suggested that salt-washing inhibits the electron transfer from the oxygen-evolving site to Z, the physiological donor to P680. In inhibited photosystem II units lacking Signal II_f, Z⁺ is rapidly reduced, possibly by a modified S-cycle unable to evolve oxygen.     

The role of a long-wavelength pigment form in the chlorophyll biosynthesis

Photoconversion of protochlorophyllide₆₅₀ form was observed in etiolated leaves illuminated with long-wavelength—690 nm—light. This process showed Shibata shift and was found to have a strong temperature dependence between 20 and –40°C. The low rate of reaction, the strong temperature dependence and calculations on the spectral overlap integral of absorption and fluorescence bands in this spectral region indicate that the phototransformation of the 650 nm form of protochlorophyllide may be caused by a back energy migration from a long-wavelength pigment form absorbing around 690 nm; this pigment form is probably a long-wavelength form of protochlorophyll/ide.     

The turnover of chlorophyll in greening wheat leaves

A method for the estimation of chlorophyll turnover in wheat leaves is presented. This is based on the inhibition of chlorophyll synthesis by treatment of the cut leaves with laevulinic acid (LA), a competitive inhibitor of δ-aminolaevulinic acid dehydratase. The turnover of chlorophyll in young, greening leaves, given short periods of light was a relatively rapid process. However, in seedlings exposed to light for longer periods the turnover became progressively slower, and was measured in days rather than hours.     

Differential regulation of the accumulation of the light-harvesting chlorophyll a/b complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves

The photoregulation of chloroplast development in pea leaves has been studied by reference to three polypeptides and their mRNAs. The polypeptides were the large subunit (LSU) and the small subunit (SSU) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO), and the light-harvesting chlorophyll a/b protein (LHCP). The polypeptides were assayed by a sensitive radioimmune assay, and the mRNAs were assayed by hybridization to cloned DNA probes. LSU, LSU mRNA, and LHCP mRNA were detectable in etiolated seedlings but LHCP, SSU, and SSU mRNA were at or below the limit of detection. During the first 48 hr of de-etiolation under continuous white light, the mRNAs for LSU, SSU, and LHCP increased in concentration per apical bud by about 40-fold, at least 200-fold, and about 25-fold, respectively, while the total RNA content per apical bud increased only 3.5-fold. In the same period, the LSU, SSU, and LHCP contents per bud increased at least 60-, 100-, and 200-fold, respectively. The LHCP increased steadily in concentration during de-etiolation, whereas the accumulation LSU, SSU, and SSU mRNA showed a 24-hr lag. The accumulation of SSU, SSU mRNA, and LHCP mRNA showed classical red/far-red reversibility, indicating the involvement of phytochrome in the regulatory mechanism. LSU and LSU mRNA were induced equally well by red and far-red light. The LHCP failed to accumulate except under continuous illumination. These results indicate that the accumulation of SSU is controlled largely through the steady-state level of its mRNA, which is in turn almost totally dependent on light as an inducer and on phytochrome as one of the photoreceptors. The accumulation of LSU is largely but not totally determined by the level of its mRNA, which appears to be under strong photoregulation, which has yet to be shown to involve phytochrome. Phytochrome is involved in the regulation of LHCP mRNA levels but substantial levels of the mRNA also occur in the dark. LHCP accumulation is not primarily governed by the levels of LHCP mRNA but by posttranslational stabilization in which chlorophyll synthesis plays a necessary but not sufficient role.