Stephen K. Herbert

Electron transport and photophosphorylation by Photosystem I in vivo in plants and cyanobacteria.

Recently, a number of techniques, some of them relatively new and many often used in combination, have given a clearer picture of the dynamic role of electron transport in Photosystem I of photosynthesis and of coupled cyclic photophosphorylation. For example, the photoacoustic technique has detected cyclic electron transport in vivo in all the major algal groups and in leaves of higher plants. Spectroscopic measurements of the Photosystem I reaction center and of the changes in light scattering associated with thylakoid membrane energization also indicate that cyclic photophosphorylation occurs in living plants and cyanobacteria, particularly under stressful conditions.In cyanobacteria, the path of cyclic electron transport has recently been proposed to include an NAD(P)H dehydrogenase, a complex that may also participate in respiratory electron transport. Photosynthesis and respiration may share common electron carriers in eukaryotes also. Chlororespiration, the uptake of O2 in the dark by chloroplasts, is inhibited by excitation of Photosystem I, which diverts electrons away from the chlororespiratory chain into the photosynthetic electron transport chain. Chlororespiration in N-starved Chlamydomonas increases ten fold over that of the control, perhaps because carbohydrates and NAD(P)H are oxidized and ATP produced by this process.The regulation of energy distribution to the photosystems and of cyclic and non-cyclic phosphorylation via state 1 to state 2 transitions may involve the cytochrome b6-f complex. An increased demand for ATP lowers the transthylakoid pH gradient, activates the b6-f complex, stimulates phosphorylation of the light-harvesting chlorophyll-protein complex of Photosystem II and decreases energy input to Photosystem II upon induction of state 2. The resulting increase in the absorption by Photosystem I favors cyclic electron flow and ATP production over linear electron flow to NADP and ‘poises’ the system by slowing down the flow of electrons originating in Photosystem II.Cyclic electron transport may function to prevent photoinhibition to the photosynthetic apparatus as well as to provide ATP. Thus, under high light intensities where CO2 can limit photosynthesis, especially when stomates are closed as a result of water stress, the proton gradient established by coupled cyclic electron transport can prevent over-reduction of the electron transport system by increasing thermal de-excitation in Photosystem II (Weis and Berry 1987). Increased cyclic photophosphorylation may also serve to drive ion uptake in nutrient-deprived cells or ion export in salt-stressed cells.There is evidence in some plants for a specialization of Photosystem I. For example, in the red alga Porphyra about one third of the total Photosystem I units are engaged in linear electron transfer from Photosystem II and the remaining two thirds of the Photosystem I units are specialized for cyclic electron flow. Other organisms show evidence of similar specialization.Improved understanding of the biological role of cyclic photophosphorylation will depend on experiments made on living cells and measurements of cyclic photophosphorylation in vivo.

Light adaptation of cyclic electron transport through Photosystem I in the cyanobacterium Synechococcus sp. PCC 7942

Photosystem I-driven cyclic electron transport was measured in intact cells of Synechococcus sp PCC 7942 grown under different light intensities using photoacoustic and spectroscopic methods. The light-saturated capacity for PS I cyclic electron transport increased relative to chlorophyll concentration, PS I concentration, and linear electron transport capacity as growth light intensity was raised. In cells grown under moderate to high light intensity, PS I cyclic electron transport was nearly insensitive to methyl viologen, indicating that the cyclic electron supply to PS I derived almost exclusively from a thylakoid dehydrogenase. In cells grown under low light intensity, PS I cyclic electron transport was partially inhibited by methyl viologen, indicating that part of the cyclic electron supply to PS I derived directly from ferredoxin. It is proposed that the increased PSI cyclic electron transport observed in cells grown under high light intensity is a response to chronic photoinhibition.

THE APPLICATION OF PHOTOACOUSTIC TECHNIQUES TO STUDIES OF PHOTOSYNTHESIS

The photoacoustic (PA)? technique is becoming an increasingly popular method in photosynthesis research. The PA effect, which is the production of sound by modulated light, was first described by Alexander Graham Bell’ but was not applied to studies of photosynthesis until the work of Malkin and Cahen.2,3 In studies of photosynthesis, the PA technique serves to quantify the conversion of absorbed light into heat in a photosynthetic sample. By difference, the fraction of absorbed light energy that is utilized in photochemistry may also be quantified. A similar use of heat measurement to quantify photosynthetic efficiency was made by Arnold in the 1 9 3 0 ~ ~ when he used the Callendar “radio balance,” an instrument designed to detect heat production by radioactive substances, to measure the small heat differences between photosynthetically active and inactive leaves, a phenomenon that had been predicted by Spoehr in 1926.5 Arnold found that intact leaves stored light energy with an efficiency of 35%, a value that he considered at that time to be far too low and that he did not publish until much later.6 Photocaustic measurements of the present day confirm that the overall efficiency of photosynthesis in intact samples is in fact, about 35%,’ a value that may also be derived theoreti ~ a l l y . ~ . ~ The growing interest in PA as applied to photosynthesis research is reflected by the number of reviews that have appeared recently and that provide an account of the progress in this field.1s2z The two previous Yearly Reviews of Balasubramanian and RaoZ3 and Braslavskyz4 appearing in this journal have summarized applications of the PA technique to the study of biological systems in general. This review limits itself to applications of this technique to studies of photosynthesis. Another review by Malkin and CanaaniZS is appearing in this year’s issue of the Annual Review of Plant Physiology.

Chloroplast iron-sulfur cluster protein maturation requires the essential cysteine desulfurase CpNifS

NifS-like proteins provide the sulfur (S) for the formation of iron-sulfur (Fe-S) clusters, an ancient and essential type of cofactor found in all three domains of life. Plants are known to contain two distinct NifS-like proteins, localized in the mitochondria (MtNifS) and the chloroplast (CpNifS). In the chloroplast, five different Fe-S cluster types are required in various proteins. These plastid Fe-S proteins are involved in a variety of biochemical pathways including photosynthetic electron transport and nitrogen and sulfur assimilation. In vitro, the chloroplastic cysteine desulfurase CpNifS can release elemental sulfur from cysteine for Fe-S cluster biogenesis in ferredoxin. However, because of the lack of a suitable mutant allele, the role of CpNifS has not been studied thus far in planta. To study the role of CpNifS in Fe-S cluster biogenesis in vivo, the gene was silenced by using an inducible RNAi (interference) approach. Plants with reduced CpNifS expression exhibited chlorosis, a disorganized chloroplast structure, and stunted growth and eventually became necrotic and died before seed set. Photosynthetic electron transport and carbon dioxide assimilation were severely impaired in the silenced plant lines. The silencing of CpNifS decreased the abundance of all chloroplastic Fe-S proteins tested, representing all five Fe-S cluster types. Mitochondrial Fe-S proteins and respiration were not affected, suggesting that mitochondrial and chloroplastic Fe-S assembly operate independently. These findings indicate that CpNifS is necessary for the maturation of all plastidic Fe-S proteins and, thus, essential for plant growth.

Changes in the cyanobacterial photosynthetic apparatus during acclimation to macronutrient deprivation.

When the cyanobacterium Synechococcus sp. Strain PCC 7942 is deprived of an essential macronutrient such as nitrogen, sulfur or phosphorus, cellular phycobiliprotein and chlorophyll contents decline. The level of β-carotene declines proportionately to chlorophyll, but the level of zeaxanthin increases relative to chlorophyll. In nitrogen- or sulfur-deprived cells there is a net degradation of phycobiliproteins. Otherwise, the declines in cellular pigmentation are due largely to the diluting effect of continued cell division after new pigment synthesis ceases and not to net pigment degradation. There was also a rapid decrease in O2 evolution when Synechococcus sp. Strain PCC 7942 was deprived of macronutrients. The rate of O2 evolution declined by more than 90% in nitrogen- or sulfur-deprived cells, and by approximately 40% in phosphorus-deprived cells. In addition, in all three cases the fluorescence emissions from Photosystem II and its antennae were reduced relative to that of Photosystem I and the remaining phycobilisomes. Furthermore, state transitions were not observed in cells deprived of sulfur or nitrogen and were greatly reduced in cells deprived of phosphorus. Photoacoustic measurements of the energy storage capacity of photosynthesis also showed that Photosystem II activity declined in nutrient-deprived cells. In contrast, energy storage by Photosystem I was unaffected, suggesting that Photosystem I-driven cyclic electron flow persisted in nutrient-deprived cells. These results indicate that in the modified photosynthetic apparatus of nutrient-deprived cells, a much larger fraction of the photosynthetic activity is driven by Photosystem I than in nutrient-replete cells.

Photoacoustic analysis indicates that chloroplast movement does not alter liquid-phase CO2 diffusion in leaves of Alocasia brisbanensis.

Light-mediated chloroplast movements are common in plants. When leaves of Alocasia brisbanensis (F.M. Bailey) Domin are exposed to dim light, mesophyll chloroplasts spread along the periclinal walls normal to the light, maximizing absorbance. Under high light, the chloroplasts move to anticlinal walls. It has been proposed that movement to the high-light position shortens the diffusion path for CO2 from the intercellular air spaces to the chloroplasts, thus reducing CO2 limitation of photosynthesis. To test this hypothesis, we used pulsed photoacoustics to measure oxygen diffusion times as a proxy for CO2 diffusion in leaf cells. We found no evidence that chloroplast movement to the high-light position enhanced gas diffusion. Times for oxygen diffusion were not shorter in leaves pretreated with white light, which induced chloroplast movement to the high-light position, compared with leaves pretreated with 500 to 700 nm light, which did not induce movement. From the oxygen diffusion time and the diffusion distance from chloroplasts to the intercellular gas space, we calculated an oxygen permeability of 2.25 × 10–6 cm2 s–1 for leaf cells at 20°C. When leaf temperature was varied from 5°C to 40°C, the permeability for oxygen increased between 5°C and 20°C but changed little between 20°C and 40°C, indicating changes in viscosity or other physical parameters of leaf cells above 20°C. Resistance for CO2 estimated from oxygen permeability was in good agreement with published values, validating photoacoustics as another way of assessing internal resistances to CO2 diffusion.

Light distribution, transfer and utilization in the marine red alga Porphyra perforata from photoacoustic energy-storage measurements

Abstract Light energy utilization in Porphyra perforata was monitored by the photoacoustic method in different conditions of illumination. Auxiliary chlorophyll a fluorescence measurements were made to estimate the fraction of open photosystem II (PS II) reaction centers. These measurements allowed a consistent quantitation of excitation distribution and transfer from PS II to PS I under the physiological conditions used. Maximum energy storage was obtained with modulated light absorbed almost exclusively by the phycobilins (light 2). Modulated light absorbed by chlorophyll a (light I) gave much smaller energy storage (about 1 3 of the maximum), which could be enhanced to the maximum by addition of background light 2. Addition of increasing intensities of background light 1 to modulated light 2 did not initially induce any effect and then decreased the energy storage to about half of the maximum. From the above results and with simple mathematical modelling, numbers were obtained for light distribution and energy transfer parameters. From the enhancement saturation curves of the effect of background light 2 on the energy storage in modulated light 1 we conclude that in state 1 light 2 is exclusively absorbed in PS II and that there is no energy transfer to PS I from open PS II reaction centers. From the value of the energy storage for light 2 in state 1 and the degree of openness of PS II reaction centers it is possible to conclude that energy transfer to PS I occurs from closed PS II reaction centers with a probability approaching 1. In state 2 light 2 is distributed more evenly (approximately in a ratio PS II PS I of 0.55:0.45) either by energy transfer via PS II from open PS II reaction centers, or by direct interactions of the phycobilins and PS I. Comparison of the maximum fluorescence values in the two states favors the second possibility. Energy transfer from PS II units with closed reaction centers occurs again in state 2 with a probability approaching 1. Comparison of energy utilization and oxygen evolution in light 1 relative to light 2 and the inhibitory effect of DCMU, which is complete in light 2 but only partial in light 1, suggests the existence of two types of PS I units: one type is engaged in electron transfer from PS II and the other type specializes in cyclic electron flow. The above quantitative analysis allows to estimate the ratio of the two types of PS I unit to be roughly about 0.3:0.7, respectively.

Acclimation of the Photosynthetic Apparatus to Growth Irradiance in a Mutant Strain of Synechococcus Lacking Iron Superoxide Dismutase.

The acclimation of the photosynthetic apparatus to growth irradiance in a mutant strain of Synechococcus sp. PCC 7942 lacking detectable iron superoxide dismutase activity was studied. The growth of the mutant was inhibited at concentrations of methyl viologen 4 orders of magnitude smaller than those required to inhibit the growth of the wild-type strain. An increased sensitivity of photosynthetic electron transport near photosystem I (PSI) toward photooxidative stress was also observed in the mutant strain. In the absence of methyl viologen, the mutant exhibited similar growth rates compared with those of the wild type, even at high growth irradiance (350 [mu]E m-2 s-1) where chronic inhibition of photosystem II (PSII) was observed in both strains. Under high growth irradiance, the ratios of PSII to PSI and of [alpha]-phycocyanin to chlorophyll a were less than one-third of the values for the wild type. In both strains, cellular contents of chlorophyll a, [alpha]-phycocyanin, and [beta]-carotene, as well as the length of the phycobilisome rods, declined with increasing growth irradiance. Only the cellular content of the carotenoid zeaxanthin seemed to be independent of growth irradiance. These results suggest an altered acclimation to growth irradiance in the sodB mutant in which the stoichiometry between PSI and PSII is adjusted to compensate for the loss of PSI efficiency occurring under high growth irradiance. Similar shortening of the phycobilisome rods in the sodB mutant and wild-type strain suggest that phycobilisome rod length is regulated independently of photosystem stoichiometry.

Cytochrome c-553 is not required for photosynthetic activity in the cyanobacterium Synechococcus.

In cyanobacteria, the water-soluble cytochrome c-553 functions as a mobile carrier of electrons between the membrane-bound cytochrome b6-f complex and P-700 reaction centers of Photosystem I. The structural gene for cytochrome c-553 (designated cytA) of the cyanobacterium Synechococcus sp. PCC 7942 was cloned, and the deduced amino acid sequence was shown to be similar to known cyanobacterial cytochrome c-553 proteins. A deletion mutant was constructed that had no detectable cytochrome c-553 based on spectral analyses and tetramethylbenzidine-hydrogen peroxide staining of proteins resolved by polyacrylamide gel electrophoresis. The mutant strain was not impaired in overall photosynthetic activity. However, this mutant exhibited a decreased efficiency of cytochrome f oxidation. These results indicate that cytochrome c-553 is not an absolute requirement for reducing Photosystem I reaction centers in Synechococcus sp. PCC 7942.

New applications of photoacoustics to the study of photosynthesis

Photoacoustic methods offer unique capabilities for photosynthesis research. Phenomena that are readily observed by photoacoustics include the storage of energy by electron transport, oxygen evolution by leaf tissue at microsecond time resolution, and the conformational changes of photosystems caused by charge separation. Despite these capabilities, photoacoustic methods have not been widely exploited in photosynthesis research. One factor that has contributed to their slow adoption is uncertainty in the interpretation of photoacoustic signals. Careful experimentation is resolving this uncertainty, however, and technical refinements of photoacoustic methods continue to be made. This review provides an overview of the application of photoacoustics to the study of photosynthesis with an emphasis on the resolution of uncertainties in the interpretation of photoacoustic signals. Recent developments in photoacoustic technology are also presented, including a microphotoacoustic spectrometer, gas permeable photoacoustic cells, the use of photoacoustics to monitor phytoplankton populations, and the use of photoacoustics to study protein dynamics.