Jan M. Anderson

Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts

The lateral distribution of the main chlorophyll-protein complexes between appressed and non-appressed thylakoid membranes has been studied. The reaction centre complexes of Photosystems I and II and the light-harvesting complex have been resolved by an SDS-polyacrylamide gel electrophoretic method which permits most of the chlorophyll to remain protein-bound. The analyses were applied to subchloroplast fractions shown to be derived from different thylakoid regions. Stroma thylakoids were separated from grana stacks by centrifugation following chloroplast disruption by press treatment or digitonin. Vesicles derived from the grana partitions were isolated by aqueous polymer two-phase partition. A substantial depletion in the amount of Photosystem I chlorophyll-protein complex and an enrichment in the Photosystem II reaction centre complex and the light-harvesting complex occurrred in the appressed grana partition region. The high enrichment in this fraction compared to grana stack fractions derived from press or digitonin-treatments, suggests that the grana Photosystem I is restricted mainly to the non-appressed grana end membranes and margins, and that the grana partitions possess mainly Photosystem II reaction centre complex and the light-harvesting complex. In contrast, stroma thylakoids are highly enriched in the Photosystem I reaction centre complex. They possess also some 10--20% of the total Photosystem II reaction centre complex and the light-harvesting complex. The ratio of light-harvesting complex to Photosystem II reaction centre complex is rather constant in all subchloroplast fractions suggesting a close association between these complexes. This was not so for the ratio of light-harvesting complex and the Photosystem I reaction centre complex. The lateral heterogeneity in the distribution of the photosystems between appressed and non-appressed membranes must have a profound impact on current understanding of both the distribution of excitation energy and photosynthetic electron transport between the photosystems.

The grand design of photosynthesis : acclimation of the photosynthetic apparatus to environmental cues

Dynamic acclimation of the photosynthetic apparatus in response to environmental cues, particularly light quantity and quality, is a widely-observed and important phenomenon which contributes to the tolerance of plants against stress and helps to maintain, as far as possible, optimal photosynthetic efficiency and resource utilization. This mini-review represents a scrutiny of a number of possible photoreceptors (including the two photosystems acting as light sensors) and signal transducers that may be involved in producing acclimation responses. We suggest that regulation by signal transduction may be effected at each of several possible points, and that there are multiple regulatory mechanisms for photosynthetic acclimation.

UV-B damage and protection at the molecular level in plants

Influx of solar UV-B radiation (280–320 nm) will probably increase in the future due to depletion of stratospheric ozone. In plants, there are several targets for the deleterious UV-B radiation, especially the chloroplast. This review summarizes the early effects and responses of low doses of UV-B at the molecular level. The DNA molecules of the plant cells are damaged by UV due to the formation of different photoproducts, such as pyrimidine dimers, which in turn can be combatted by specialized photoreactivating enzyme systems. In the chloroplast, the integrity of the thylakoid membrane seems to be much more sensitive than the activities of the photosynthetic components bound within. However, the decrease of mRNA transcripts for the photosynthetic complexes and other chloroplast proteins are among very early events of UV-B damage, as well as protein synthesis. Other genes, encoding defence-related enzymes, e.g., of the flavonoid biosynthetic pathway, are rapidly up-regulated after commencement of UV-B exposure. Some of the cis-acting nucleotide elements and trans-acting protein factors needed to regulate the UV-induced expression of the parsley chalcone synthase gene are known.

Photoinhibition of photosynthesis represents a mechanism for the long-term regulation of photosystem II

The obligate shade plant, Tradescantia albiflora Kunth grown at 50 μmol photons · m−2 s−1 and Pisum sativum L. acclimated to two photon fluence rates, 50 and 300 μmol · m−2 · s−1, were exposed to photoinhibitory light conditions of 1700 μmol · m−2 · s−1 for 4 h at 22° C. Photosynthesis was assayed by measurement of CO2-saturated O2 evolution, and photosystem II (PSII) was assayed using modulated chlorophyll fluorescence and flash-yield determinations of functional reaction centres. Tradescantia was most sensitive to photoinhibition, while pea grown at 300 μmol · m−2 · s−1 was most resistant, with pea grown at 50 μmol · m−2 · s−1 showing an intermediate sensitivity. A very good correlation was found between the decrease of functional PSII reaction centres and both the inhibition of photosynthesis and PSII photochemistry. Photoinhibition caused a decline in the maximum quantum yield for PSII electron transport as determined by the product of photochemical quenching (qp) and the yield of open PSII reaction centres as given by the steady-state fluorescence ratio, F′vF′m, according to Genty et al. (1989, Biochim. Biophys. Acta 990, 81–92). The decrease in the quantum yield for PSII electron transport was fully accounted for by a decrease in F′vF′m, since qp at a given photon fluence rate was similar for photoinhibited and noninhibited plants. Under lightsaturating conditions, the quantum yield of PSII electron transport was similar in photoinhibited and noninhibited plants. The data give support for the view that photoinhibition of the reaction centres of PSII represents a stable, long-term, down-regulation of photochemistry, which occurs in plants under sustained high-light conditions, and replaces part of the regulation usually exerted by the transthylakoid ΔpH gradient. Furthermore, by investigating the susceptibility of differently lightacclimated sun and shade species to photoinhibition in relation to qp, i.e. the fraction of open-to-closed PSII reaction centres, we also show that irrespective of light acclimation, plants become susceptible to photoinhibition when the majority of their PSII reaction centres are still open (i.e. primary quinone acceptor oxidized). Photoinhibition appears to be an unavoidable consequence of PSII function when light causes sustained closure of more than 40% of PSII reaction centres.

Fractionation of the photochemical systems of photosynthesis I. Chlorophyll contents and photochemical activities of particles isolated from spinach chloroplasts

Spinach chloroplasts were fragmented by incubation with digitonin and the resulting chlorophyll-containing particles separated by differential centrifugation. Particles pelleted by forces of 1000 × g and 10 000 × g had a lower ratio of chlorophyll a/chlorophyll b (chl a/chl b) than the chloroplasts, whereas the smaller particles which either sedimented at 50 000–144 000 × g or remained in the 144 000 × g supernatant had higher ratios than the chloroplasts. Chl a/chl b ratios were usually determined spectrophotometrically in 80% acetone, but essentially the same ratios were obtained after transfer of the pigments to ether or after separation of the chlorophylls by thin-layer chromatography. The small particles were inactive in the Hill reaction, either with ferricyanide, trichlorophenolindophenol or NADP+ as oxidant, but they photoreduced NADP+ if provided with the electron-donor couple, sodium ascorbate and dichlorophenolindophenol, and both ferredoxin and NADP reductase. The 1000 × g and 10 000 × g fractions showed Hill activity, but the rate of reduction of NADP+ in the presence of ferredoxin and NADP reductase was lower than the rates of reduction of ferricyanide and trichlorophenolindophenol. The particles with the high chl a/chl b ratios appear to be representative of System 1 or the “long wavelength” system of photosynthesis, whereas the 1000 × g and 10 000 × g fractions appear to be enriched in particles representative of System 2. Chloroplasts were also fragmented by Triton X-100 and Nonidet P-40; in contrast to the digitonin treatment, the particles so obtained were photochemically inactive.

Effects of supplementary ultraviolet-B radiation on photosynthesis in Pisum sativum

Pea plants (Pisum sativum L., cv. Greenfeast) were exposed to supplementary UV-B light (up to 8 days) starting on the 17th day after sowing. The effects of this exposure on photosynthesis and the content and activities of some chloroplast components of the mature leaves of these plants were studied. (i) The total chorophyll content of pea leaves was approximately 40% of that in the control leaves on the 8th day of UV-B exposure. Chlorophyll a levels decreased to a greater extent than the content of chlorophyll b. The decrease in carotenoids paralleled the decrease in chlorophyll b. (ii) On a chlorophyll basis, the contents of Photosystem I and cytochrome f were stable, whereas Photosystem II, ATP hydrolysis by the ATP synthase and the maximum ribulose-1,5-bisphosphate carboxylase (Rubisco) activity decreased by 55, 47 and 80%, respectively, when compared with the controls at the end of the 8-day illumination period. (iii) On a leaf-area basis, Photosystem I and cytochrome f content decreased by 58%, Photosystem II by 80%, ATP hydrolysis by 80%, and Rubisco activity by 90%, when compared with the controls. The in vivo activation of Rubisco was markedly increased in UV-B-treated pea leaves. The underlying mechanisms for these results are discussed.

Photoinhibition and D1 Protein Degradation in Peas Acclimated to Different Growth Irradiances

The relationship between the susceptibility of photosystem II (PSII) to photoinhibition in vivo and the rate of degradation of the D1 protein of the PSII reaction center heterodimer was investigated in leaves from pea plants (Pisum sativum L. cv Greenfeast) grown under widely contrasting irradiances. There was an inverse linear relationship between the extent of photoinhibition and chlorophyll (Chl) a/b ratios, with low-light leaves being more susceptible to high light. In the presence of the chloroplast-encoded protein synthesis inhibitor lincomycin, the differential sensitivity of the various light-acclimated pea leaves to photoinhibition was largely removed, demonstrating the importance of D1 protein turnover as the most crucial mechanism to protect against photoinhibition. In the differently light-acclimated pea leaves, the rate of D1 protein degradation (measured from [35S]methionine pulse-chase experiments) increased with increasing incident light intensities only if the light was not high enough to cause photoinhibition in vivo. Under moderate illumination, the rate constant for D1 protein degradation corresponded to the rate constant for photoinhibition in the presence of lincomycin, demonstrating a balance between photodamage to D1 protein and subsequent recovery, via D1 protein degradation, de novo synthesis of precursor D1 protein, and reassembly of functional PSII. In marked contrast, in light sufficiently high to cause photoinhibition in vivo, the rate of D1 protein degradation no longer increased concomitantly with increasing photoinhibition, suggesting that the rate of D1 protein degradation is playing a regulatory role. The extent of thylakoid stacking, indicated by the Chl a/b ratios of the differently light-acclimated pea leaves, was linearly related to the half-life of the D1 protein in strong light. We conclude that photoinhibition in vivo occurs under conditions in which the rate of D1 protein degradation can no longer be enhanced to rapidly remove irreversibly damaged D1 protein. We suggest that low-light pea leaves, with more stacked membranes and less stroma-exposed thylakoids, are more susceptible to photoinhibition in vivo mainly due to their slower rate of D1 protein degradation under sustained high light and their slower repair cycle of the photodamaged PSII centers.

Consequences of spatial separation of photosystem 1 and 2 in thylakoid membranes of higher plant chloroplasts

The thylakoid membranes of most higher plants and some green algae are structurally organized into a network of closely contacting, appressed membranes, the grana thylakoids, which are interconnected with single, unstacked membranes, the stroma thylakoids [l-5]. The inner surface of these thylakoid membranes encloses a space which is continuous between the grana and stroma thylakoids. As shown schematically in fig.1, thylakoids have two distinct membrane regions, termed here exposed and appressed membranes. The exposed ~ylakoids whose outer surfaces are in direct contact with the stroma, include stroma ~ylakoids and the end membr~es and margins of the grana stacks. In contrast, the outer surfaces of the appressed membranes of the grana partitions have limited access to the stroma. Freeze-fracture electron microscopy reveals a difference in the size, shape and density of freeze-fracture particles located in appressed and exposed membranes [5-81. This reflects a difference in the distribution of the main intrinsic macromolecular complexes of thylakoid membranes in the two regions. This striking structural organization of thylakoids is paralleled by a differentiation of function. Fractionation of ~ylakoids into grana and stroma thylakoid fractions by detergent [9] or mechanical methods [IO], shows that the large subchlo-

The dynamic photosynthetic membrane and regulation of solar energy conversion.

Abstract The photosynthetic apparatus of higher plants is remarkably adaptable both to sudden stress conditions and to longer-term changes in light intensity. Clues to the molecular mechanisms involved in the intricate regulatory networks highlight the extraordinarily dynamic nature of the photosynthetic membranes. An intriguing lateral migration of certain proteins and protein complexes between the appressed and nonappressed regions of the membrane maintains a lateral heterogeneity of function between these two regions to optimize photosynthesis and minimize damage to the photosystems.

Mechanistic differences in photoinhibition of sun and shade plants

Leaf discs of the shade plant Tradescantia albiflora Kunth grown at 50 μmol · m−2 · s−1, and the facultative sun/shade plant Pisum sativum L. grown at 50 or 300 μmol · m−2, s−1, were photoinhibited for 4 h in 1700 μmol photons m−2 · s−1 at 22° C. The effects of photoinhibition on the following parameters were studied: i) photosystem II (PSII) function; ii) amount of D1 protein in the PSII reaction centre; iii) dependence of photoinhibition and its recovery on chloroplast-encoded protein synthesis; and, iv) the sensitivity of photosynthesis to photoinhibition in the presence or absence of the carotenoid zeaxanthin. We show that: i) despite different sensitivities to photoinhibition, photoinhibition in all three plants occurred at the reaction centre of PSII; ii) there was no correlation between the extent of photoinhibition and the degradation of the D1 protein; iii) the susceptibility to photoinhibition by blockage of chloroplas-tencoded protein synthesis was much less in shade plants than in plants acclimated to higher light; and iv) inhibition of zeaxanthin formation increased the sensitivity to photoinhibition in pea, but not in the shade plant Tradescantia. We suggest that there are mechanistic differences in photoinhibition of sun and shade plants. In sun plants, an active repair cycle of PSII replaces photoinhibited reaction centres with photochemically active ones, thereby conferring partial protection against photoinhibition. However, in shade plants, this repair cycle is less important for protection against photoinhibition; instead, photoinhibited PSII reaction centres may confer, as they accumulate, increased protection of the remaining connected, functional PSII centres by controlled, nonphotochemical dissipation of excess excitation energy.