Geoffrey Hind

Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell‐to‐cell movement

Virus‐encoded movement protein (MP) mediates cell‐to‐cell spread of tobacco mosaic virus (TMV) through plant intercellular connections, the plasmodesmata. The molecular pathway by which TMV MP interacts with the host cell is largely unknown. To understand this process better, a cell wall‐associated protein that specifically binds the viral MP was purified from tobacco leaf cell walls and identified as pectin methylesterase (PME). In addition to TMV MP, PME is recognized by MPs of turnip vein clearing virus (TVCV) and cauliflower mosaic virus (CaMV). The use of amino acid deletion mutants of TMV MP showed that its domain was necessary and sufficient for association with PME. Deletion of the PME‐binding region resulted in inactivation of TMV cell‐to‐cell movement.

The role of chloride ion in photosynthesis III. The effect of artificial electron donors upon electron transport

4- to more than 10-fold acceleration of the Hill reaction (electron acceptor: indophenol dyes, ferricyanide, FMN, etc.) by Cl− is consistently observed over a wide range of pH (5.7–8.3) when EDTA-uncoupled chloroplasts are used as experimental material. Using such highly Cl−-sensitive chloroplasts the effect of Cl− was examined on the photooxidation of reducing agents which are capable of donating electrons to Photosystem II. No Cl− effect is observed on the photooxidation of hydroxylamine (at substrate level with indophenol dye as electron acceptor) or of ascorbate (FMN as acceptor). These observations strongly suggest that Cl− acts near the water-splitting end of Photosystem II. High concentrations of ammonia and methylamine, like low concentrations of hydroxylamine, block water splitting on the oxidizing side of Photosystem II. Apparently the unprotonated forms of simple amines act as a specific inhibitor at a point near the site of Cl− involvement. Upon removal of Cl− the steady-state fluorescence yield of chloroplasts is markedly and reversibly depressed. However, the addition of a substrate level of hydroxylamine obliterates the Cl− effect on fluorescence and the yield becomes higher than in the presence of Cl− alone. These observations are discussed in connection with the site of Cl− involvement and with the current model of the chloroplast fluorescence quenching mechanism.

The role of Cl− in photosynthesis I. The Cl− requirement of electron transport

Abstract 1. 1. Non-cyclic electron flow in isolated chloroplasts at high pH is dependent on Cl − or on some other monovalent anion derived from an inorganic acid with ionization constant of approx. 0.01. 2. 2. Whole chloroplasts generally do not show a Cl − requirement until the internal Cl − is released by some treatment to damage the outer membrane and promote swelling. 3. 3. The basal component of non-cyclic electron flow is much less dependent on Cl − than the uncoupled or coupled components. Cl − effects have been observed after uncoupling by ammonia, chloroquine and carbonyl cyanide m -chlorophenylhydrazone. 4. 4. The Cl − effect is associated closely with a photochemical reaction (Photosystem II). 5. 5. Photoinactivation in red light is potentiated by Cl − deficiency, but is not in itself the cause of the Cl − effect. 6. 6. The role of Cl − is discussed with reference to the chemi-osmotic coupling hypothesis.

ORGANIZATION OF PIGMENT‐PROTEIN COMPLEXES INTO MACRODOMAINS IN THE THYLAKOID MEMBRANES OF WILD‐TYPE and CHLOROPHYLL fo‐LESS MUTANT OF BARLEY AS REVEALED BY CIRCULAR DICHROISM

The organization of pigment‐protein complexes into large chiral macrodomains was investigated in wild‐type and chlorophyll b‐less mutant thylakoid membranes of barley. The variations in the anomalous circular dichroism bands and in the angular‐dependence of circular intensity differential scattering showed that in wild‐type chloroplasts, the formation of macrodomains was governed by interactions of the light‐harvesting chlorophyll alb complexes (LHCII). Two external factors could be identified which regulate the parameters of the anomalous circular dichroism signal: (i) electrostatic screening by divalent cations under conditions that favor membrane stacking and (ii) the osmotic pressure of the medium, which is suggested to affect the lateral interactions between complexes and influence the packing‐density of particles. These two factors governed preferentially the negative and the positive anomalous circular dichroism signals, respectively. In the chlorina f‐2 mutant thylakoid membranes, deficient in most chlorophyll b binding proteins, the formation of macrodomains which gave rise to the anomalous circular dichroism signals was still regulated by these same external factors. However, in the absence of major LHCII polypeptides the formation of macrodomains was apparently mediated by other complexes having weaker interaction capabilities. As a consequence, the size of the macrodomains under comparable conditions appeared smaller in the mutant than in the wild‐type thylakoid membranes.

Partial characterization of cyclic electron transport in intact chloroplasts

Abstract Turnover of the cyclic electron transfer chain around photosystem I in intact chloroplasts was induced by addition of sodium dithionite after poisoning with 3-(3,4-dichlorophenyl)-1,1-dimethylurea. A substantial permeability barrier to dithionite allowed redox poising to a level sufficiently negative to activate, but not overreduce, the cycle. Spectral changes could thus be studied without interference from photosystem II reactions. Illumination by repetitive single-turnover flashes showed the participation in the cycle of cytochromes f and b 563 with an apparent 1:1 stoichiometry. The rise of the flash-induced electrochromic bandshift (“P518”) showed a fast phase with rise time b 563 and f was uninhibited. A kinetic correlation was observed between the rise of the slow phase and the rereduction of cytochrome f , whereas cytochrome b 563 reoxidation was slower than both. Redox titrations of the appearance of the slow rise in the P518 response showed that it was only observed on repetitive flashes when a component of midpoint potential ∼- −55 mV (pH 8.1), n = 2, was reduced before the flash. A comparison is drawn between this protonmotive electron transfer cycle and that of the purple nonsulfur bacterium Rhodopseudomonas capsulata ; possible arrangements of electron carriers in the photosystem I cycle are discussed, and a modified Q cycle is proposed to account for the properties observed.

Electron transport pathways in spinach chloroplasts. Reduction of the primary acceptor of Photosystem II by reduced nicotinamide adenine dinucleotide phosphate in the dark

Addition of NADPH to osmotically lysed spinach chloroplasts results in a reduction of the primary acceptor (Q) of photosystem II. This reduction of Q reaches a maximum of 50% in chloroplasts maintained under weak illumination and requires added ferredoxin and Mg2+. The reaction is inhibited by (I) an antibody to ferredoxin-NADP+ reductases (EC 1.6.7.1), (ii) treatment of chloroplasts with N-ethylmaleimide in the presence of NADPH, (iii) disulfodisalicylidenepropanediamine, (iv) antimycin, and (v) acceptors of non-cyclic electron transport. Uncouplers of phosphorylation do not affect NADPH-driven reduction of Q. It is proposed that electron flow from NADPH to Q may occur in the dark by a pathway utilising portions of the normal cyclic and non-cyclic electron carrier sequences. The possible in vivo role for such a pathway in redox poising of cyclic electron transport and hence in controlling the ATP/NADPH supply ratio is discussed.

The involvement of ferredoxin-NADP+ reductase in cyclic electron transport in chloroplasts

The sites of action, in spinach thylakoid, of known inhibitors of electron transport at the reducing end of photosystem I have been more accurately located by parallel investigation of effects on three partial reactions: photo-reduction (from water) of added NADP+, photoreduction of added cytochrome c, and dark reduction of cyto-chrome c by added NADPH. Comparison with inhibitory effects on cyclic electron flow (registered by the slow phase of the electrochromic response during repetitive flash excitation) permitted assessment of the role of ferredoxin and ferredoxin-NADP+ reductase (ferredoxin: NADP+ oxidoreductase, EC 1.18.1.3) in the cyclic electron transport pathway around photosystem I. Disulfodisalicylidenepropane-1,1-diamine inhibited all the above partial reactions except the ferredoxin-dependent photoreduction of cytochrome C. thereby indicating its interference with the reductase or the complexation between reductase and ferredoxin. Studies with purified ferredoxin-NADP+ reductase established it as the sensitive component. Cyclic flow is also sensitive to the above inhibitor and thus presumably involves the reductase. Supporting evidence for this came from studies of inhibition by substituted maleimides, which are inhibitors of electron transfer through the isolated reductase; these also inhibited the slow phase of the electrochromic response and all partial reactions except the photoreduction of cytochrome c. In contrast, an antiserum against the reductase affected only reactions involving NADP. The conclusion is drawn that the pathway of cyclic electron transport includes both ferredoxin and ferredoxin-NADP+ reductase, but not the NADP-binding site on the reductase.

Association of ferredoxin-NADP+ oxidoreductase with the chloroplast cytochrome b-f complex

The 37‐kDa non‐heme component in spinach cytochrome b‐f complex prepared from EDTA‐washed thylakoids [(1983) J. Biol. Chem. 258, 10348‐10354] is shown to be ferredoxin‐NADP+ oxidoreductase (EC 1.18.1.2) on the basis of immunoreactivity, amino acid analysis, and pattern of cleavage by cyanogen bromide. Strong binding of the reductase to the isolated cytochrome complex suggests this is an important site for its attachment to the thylakoid membrane in vivo.

The kinetics of the pH rise in illuminated chloroplast suspensions.

A flow method applied to a pH-measurement system was able to resolve the initial phase (<1 sec) of the kinetics of pH changes in illuminated chloroplast suspensions. Immediately upon illumination, a rapid pH rise takes place which stops abruptly when the light is turned off; there is no significant post-illumination pH rise (‘overshoot’). The marked overshoot phenomenon observed in a conventional pH recording system was analyzed and shown to be due to an instrument response lag. A graphical treatment is suggested by which ordinary pH change curves can be corrected to remove instrumental artefacts. Flash yield determinations revealed that the initial kinetics of the pH rise are biphasic. The first, rapid phase is selectively suppressed by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and by 2,6-dichlorophenol-indophenol. The maximal slope of the second, exponential phase of pH rise with pyocyanine, FMN and ferricyanide as electron acceptors corresponded to about 400, 150 and 100 μequiv H+h per mg chlorophyll, respectively (pH 6.2, 5°). Simultaneous measurement of electron transport and pH rise indicated that the maximum stoichiometry of H+ uptake, the H+e2− ratio, may be 4.0.

Cyclic electron transport in isolated intact chloroplasts. Further studies with antimycin

Antimycin has been used to study the role of cyclic electron transport in isolated intact chloroplasts maintained under aerobic conditions. At all light intensities, antimycin inhibits CO2 fixation when assay conditions are optimal. When turnover of the Calvin cycle is inhibited, antimycin stimulates bicarbonate-dependent O2 evolution. Energy-dependent processes such as chlorophyll a and 9-aminoacridine fluorescence quenching, and light-scattering (apparent absorption) changes are inhibited by antimycin. The results suggest that cyclic electron transport contributes to photophosphorylation under aerobic conditions and is obligatory as a source of ATP during the most active periods of CO2 fixation in vivo. Cyclic electron transport can be stimulated either by inhibiting Photosystem II activity or increasing the turnover of Photosystem I relative to Photosystem II. These effects are interpreted in terms of the need for correct redox poising of carriers in the pathway in order to sustain maximum rates of cyclic electron flow. Binding studies indicate the presence of a high affinity antimycin binding site on chloroplast membranes. The stoichiometry and dissociation constant of the high affinity site are consistent with the idea that antimycin inhibits cyclic electron transport by binding to a b-type cytochrome in the thylakoid membrane.