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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.

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;Evolution of Photosynthesis' (1970), re-examined thirty years later.

I have re-examined my 1970 article 'Evolution of Photosynthesis' (Olson JM, Science 168: 438-446) to see whether any of my original proposals still survive. My original conviction that the evolution of photosynthesis was intimately connected with the origin of life has been replaced with the realization that photosynthesis may have been invented by the Bacteria after their divergence from the Archea. The common ancestor of all extant photosynthetic bacteria and cyanobacteria probably contained bacteriochlorophyll a, rather than chlorophyll a as originally proposed, and may have carried out CO(2) fixation instead of photoassimilation. The first electron donors were probably reduced sulfur compounds and later ferrous iron. The common ancestor of all extant reaction centers was probably similar to the homodimeric RC1 of present-day green sulfur bacteria (Chlorobiaceae) and heliobacteria. In the common ancestor of proteobacteria and cyanobacteria, the gene for the primordial RC1 was apparently duplicated and one copy split into two genes, one for RC2 and the other for a chlorophyll protein similar to CP43 and CP47 in extant cyanobacteria and chloroplasts. Homodimeric RC1 and homodimeric RC2 functioned in series as in the Z-scheme to deliver electrons from Fe(OH)(+) to NADP(+), while RC1 and/or RC2 separately drove cyclic electron flow for the production of ATP. In the line of evolution leading to proteobacteria, RC1 and the chlorophyll protein were lost, but RC2 was retained and became heterodimeric. In the line leading to cyanobacteria, both RC1 and RC2 replaced bacteriochlorophyll a with chlorophyll a and became heterodimeric. Heterodimeric RC2 further coevolved with a Mn-containing complex to utilize water as the electron donor for CO(2) fixation. The chlorophyll-protein was also retained and evolved into CP43 and CP47. Heliobacteria are the nearest photosynthetic relatives of cyanobacteria. The branching order of photosynthetic genes appears to be (1) proteobacteria, (2) green bacteria (Chlorobiaceae plus Chloroflexaceae), and (3) heliobacteria plus cyanobacteria.

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History of the word photosynthesis and evolution of its definition.

In 1893, Charles Barnes (1858-1910) proposed that the biological process for 'synthesis of complex carbon compounds out of carbonic acid, in the presence of chlorophyll, under the influence of light' should be designated as either 'photosyntax' or 'photosynthesis.' He preferred the word 'photosyntax,' but 'photosynthesis' came into common usage as the term of choice. Later discovery of anoxygenic photosynthetic bacteria and photophosphorylation necessitated redefinition of the term. This essay examines the history of changes in the meaning of photosynthesis.

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The Laboratory of Photosynthesis and its successors at Gif-sur-Yvette, France.

This article is not a survey of all the research made during the last half century at the 'Laboratoire de Photosynthèse' of the 'Centre National de la Recherche Scientifique' (CNRS) in Gif-sur-Yvette, but rather some personal recollections, as faithful as possible. Not all people could be mentioned and the scientists named here are mainly those who, at different stages of the laboratory's evolution, created their research teams, within or outside the laboratory. The laboratory, closed now as an administrative entity, was founded in 1953 by the CNRS in Gif-sur-Yvette, near Paris. Besides the emerging research groups in Paris and at Saclay, it was then the only one in France to be entirely dedicated to photosynthesis. Initially, the focus was on metabolic biochemistry of photosynthesis in whole plants and unicellular algae. In 1959, biophysics of primary and associated processes was added and in 1966, the laboratory was enlarged to include molecular genetics and, somewhat later, structural biology. Most of the early members of the laboratory have now gone offstage, but the research goes on, in Gif and elsewhere, thanks to the numerous high-level scientists that have been trained there. Most of the basic questions have now been answered, and interest has shifted in two directions, atomic and integrated, while many other facets of research are no longer specific to photosynthesis but part of more general biological problems, a normal situation for an area that has reached its maturity.

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The action of a range of supplementary ultraviolet (UV) wavelengths on photosynthesis in Brassica napus L. in the natural environment: effects on PS II, CO(2) assimilation and level of chloroplast proteins.

The effects of different wavebands of UV radiation on photosynthesis and the expression and abundance of photosynthetic proteins in oilseed rape (Brassica napus L. cv. Rebel) were investigated. Plants were grown outdoors under natural radiation (52 degrees N, 0 degrees E) supplemented with six wavebands of UV radiation (0.4 Wm(-2)) between 313 nm and 356 nm. A control treatment was centred at 343 nm. Exposure to supplementary UV-A radiation (320-400 nm) had no significant effects, however UV-B radiation, centred at 313 nm, caused a marked reduction in photosynthesis. This decrease was related to a reduction in the initial carboxylation velocity of Rubisco which was further correlated with a large reduction in the expression and abundance of both large and small subunits of Rubisco. These results indicate a molecular mechanism behind UV-B induced reductions in photosynthesis per unit area in plants grown under field conditions.

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Pictorial demonstrations of photosynthesis.

Theodor Engelmann's experiments in 1882 provided the first recorded visual demonstration of light wavelengths that are absorbed by photosynthetic pigments. Later, starch images in intact leaves were used to demonstrate photosynthesis in green plants. Similarly, light-induced chloroplast movements can form images in leaves as a result of changes in light transmittance through leaves and photoinhibition can form images that can be visualized by whole leaf chlorophyll fluorescence. This paper provides a brief account of how photosynthesis has been used to create an assortment of 'living images' that offer stunning demonstrations of various aspects of photosynthesis.

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Thinking about the evolution of photosynthesis.

Photosynthesis is an ancient process on Earth. Chemical evidence and recent fossil finds indicate that cyanobacteria existed 2.5-2.6 billion years (Ga) ago, and these were certainly preceded by a variety of forms of anoxygenic photosynthetic bacteria. Carbon isotope data suggest autotrophic carbon fixation was taking place at least a billion years earlier. However, the nature of the earliest photosynthetic organisms is not well understood. The major elements of the photosynthetic apparatus are the reaction centers, antenna complexes, electron transfer complexes and carbon fixation machinery. These parts almost certainly have not had the same evolutionary history in all organisms, so that the photosynthetic apparatus is best viewed as a mosaic made up of a number of substructures each with its own unique evolutionary history. There are two schools of thought concerning the origin of reaction centers and photosynthesis. One school pictures the evolution of reaction centers beginning in the prebiotic phase while the other school sees reaction centers evolving later from cytochrome b in bacteria. Two models have been put forth for the subsequent evolution of reaction centers in proteobacteria, green filamentous (non-sulfur) bacteria, cyanobacteria, heliobacteria and green sulfur bacteria. In the selective loss model the most recent common ancestor of all subsequent photosynthetic systems is postulated to have contained both RC1 and RC2. The evolution of reaction centers in proteobacteria and green filamentous bacteria resulted from the loss of RC1, while the evolution of reaction centers in heliobacteria and green sulfur bacteria resulted from the loss of RC2. Both RC1 and RC2 were retained in the cyanobacteria. In the fusion model the most recent common ancestor is postulated to have given rise to two lines, one containing RC1 and the other containing RC2. The RC1 line gave rise to the reaction centers of heliobacteria and green sulfur bacteria, and the RC2 line led to the reaction centers of proteobacteria and green filamentous bacteria. The two reaction centers of cyanobacteria were the result of a genetic fusion of an organism containing RC1 and an organism containing RC2. The evolutionary histories of the various classes of antenna/light-harvesting complexes appear to be completely independent. The transition from anoxygenic to oxygenic photosynthesis took place when the cyanobacteria learned how to use water as an electron donor for carbon dioxide reduction. Before that time hydrogen peroxide may have served as a transitional donor, and before that, ferrous iron may have been the original source of reducing power.

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Contrasting responses of photosynthesis and carbon metabolism to low temperatures in tall fescue and clovers.

Growth, photosynthesis and carbohydrate metabolism in plants of two grassland species, clover (Trifolium subterraneum L. cv. Areces and Gaitan) and tall fescue (Festuca arundinacea Schreb.), shifted from 25 to 12 degrees C for 1 day or developed at 12 degrees C were compared with controls kept at 25 degrees C. Cold development produced a larger inhibition of growth in fescue than in clovers. In contrast, transferring plants from high to low temperature inhibited photosynthesis to a lesser extent in fescue than in clovers, this difference being associated with an increase in the activation state of Calvin cycle enzymes in fescue, but not in the clovers, a decreased cytosolic fructose-1,6-bisphosphatase (cFBPase, EC 3.1.3.11) activity in clovers, and an accumulation of hexose phosphates only in fescue. Development at 12 degrees C partly relieved the inhibition of photosynthesis in clovers, in contrast with fescue, which correlated with increases in total ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco, EC 4.1.1.39) activity only in clovers, and with greater increases in total stromal FBPase (sFBPase) activity in clovers than in fescue. The activity of sucrose synthesis enzymes was increased in the two clovers and fescue developed in the cold, while carbohydrate accumulation was much bigger in cold-developed fescue than in clovers because of a 5-fold increase in fructan contents in the former. The contents of phosphorylated intermediates increased in clovers but decreased in fescue grown at 12 degrees C. Our results suggest that restricted ribulose-1,5-bisphosphate (RuBP) regeneration limited the recovery of photosynthetic capacity in cold-developed fescue.

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Light limitation of photosynthesis and activation of ribulose bisphosphate carboxylase in wheat seedlings.

In limiting light the activation of ribulose-1,5-bisphosphate (RuP(2)) carboxylase [3-phospho-D-glycerate carboxylyase (dimerizing), EC 4.1.1.39] in leaf extracts of 7- to 8-day-old wheat seedlings changed proportionally with the photosynthetic rate of the intact plants. Higher rates of photosynthesis, induced by increasing irradiances, were accompanied by an increase in activation of the leaf RuP(2) carboxylase, while RuP(2) levels remained unchanged. The degree of activation varied from 20% to 60% of full activation at irradiances of 225-1650 muE/m(2).s (photosynthetically active radiation; E = einstein, 1 mol of photons). Between 225 muE/m(2).s and darkness, activation approached 50% while RuP(2) levels dropped more than 90%. During steady-state photosynthesis, levels of the substrate RuP(2) were 250-300 nmol/mg of chlorophyll in the leaves and were similar at all irradiances above 225 muE/m(2).s (25% of light saturation). When velocities of the carboxylase in leaf extracts were corrected for CO(2) levels estimated to exist within the leaf, they compared favorably with the photosynthetic rates of the intact seedlings. Comparison of CO(2) exchange rate, RuP(2) level, and activation of the carboxylase indicates that light limitation of photosynthesis can be due to two factors: the availability of RuP(2) in dark to dim light and activation of the RuP(2) carboxylase in dim light and higher irradiances.

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Insight into the relationship of chlorophyll a fluorescence yield to the concentration of its natural quenchers in oxygenic photosynthesis.

Fluorescence of chlorophyll a (Chla) is a noninvasive and very sensitive intrinsic probe of photosynthesis. It monitors the composition and organization of the photosystems, the exciton energy transfer, the photochemistry, and the effects of various types of stress on plants. It is the most used as well as the most abused tool in photosynthesis. Thus, an understanding of its relationship to photosynthesis has been of paramount importance. Both the oxidized primary plastoquinone, QA, and the oxidized primary reaction-center Chla, P680+ (for short, P+), are known to be quenchers of Chla fluorescence yield (phi f) of photosystem II. Flash-number dependence of Chla fluorescence yield shows either a period 4, due to the four-step charge-accumulation process of water oxidation (donor side), or period 2 behavior, due to the two-step reduction of the plastoquinone QB (acceptor side) of photosystem II reaction centers. We provide here a further insight into the relationship of variable Chla fluorescence yield (phi f) to the concentration of the two quenchers. The observed time dependence of the ratio of psi f after flash 3 to that after flash 1 (or flash 5) in spinach thylakoids at pH 6 can be explained if we suggest that 1/psi f approximately equals a[PQA] + c, where a, b, and c are constants. From this it follows that the quenching of Chla fluorescence by P680+ after a flash is dependent on QA: for low [QA] (when most reaction centers are closed, [PQA] is low) the quenching of Chla fluorescence by P680+ predominates, while for high [QA] (when most reaction centers are open), the quenching of Chla fluorescence is due predominantly to the increased concentration of the reduced form of P680 ([P+] is low).

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A photosystem I reaction center driven by chlorophyll d in oxygenic photosynthesis.

A far-red type of oxygenic photosynthesis was discovered in Acaryochloris marina, a recently found marine prokaryote that produces an atypical pigment chlorophyll d (Chl d). The purified photosystem I reaction center complex of A. marina contained 180 Chl d per 1 Chl a with PsaA-F, -L, -K, and two extra polypeptides. Laser excitation induced absorption changes of reaction center Chl d that was named P740 after its peak wavelength. A midpoint oxidation reduction potential of P740 was determined to be +335 mV. P740 uses light of significantly low quantum energy (740 nm = 1.68 eV) but generates a reducing power almost equivalent to that produced by a special pair of Chl a (P700) that absorbs red light at 700 nm (1.77 eV) in photosystem I of plants and cyanobacteria. The oxygenic photosynthesis based on Chl d might either be an acclimation to the far-red light environments or an evolutionary intermediate between the red-absorbing oxygenic and the far-red absorbing anoxygenic photosynthesis that uses bacteriochlorophylls.

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Effects of atmospheric humidity and acclimation temperature on the temperature response of photosynthesis in young Larix decidua Mill.

Larch (Larix decidua Mill.) seedlings of a low altitude (600 m) Austrian provenance were raised outdoors and acclimated in chambers for 14 to 24 days during August and September at either 8 degrees C and an atmospheric saturation vapor pressure deficit (DeltaW) of 2.5 Pa kPa(-1), or 24 degrees C and a DeltaW of 6.2 Pa kPa(-1). Subsequently, their rates of photosynthesis, dark respiration and transpiration were measured at temperatures between 5 and 30 degrees C with DeltaW either maintained below 10 Pa kPa(-1) or allowed to increase with temperature up to 38 Pa kPa(-1). Below 15 degrees C the photosynthetic rate of cold-acclimated plants was higher, but above 15 degrees C it was lower, than that of warm-acclimated plants. Temperature acclimation caused a greater shift in the temperature optimum for photosynthesis when DeltaW was kept small than when it was allowed to increase with temperature. When DeltaW was kept small, leaf conductance of cold-acclimated plants, unlike that of warm-acclimated plants, did not increase with temperature above 15 degrees C. When DeltaW increased with temperature, leaf conductance of cold-acclimated plants decreased more rapidly with temperature than that of warm-acclimated plants. Low temperature acclimation increased the rate of photosynthesis below 15 degrees C without affecting leaf conductance, which indicates that there was an adaptation in leaf internal processes. Further evidence of a metabolic adaptation to acclimation temperature is that dark respiration of cold-acclimated plants was twice that of warm-acclimated plants at all temperatures.

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Effects of foliar nitrogen concentration on photosynthesis and water use efficiency in Douglas-fir.

Leaf-level physiological processes were studied in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) to determine whether apparent increases in stand-level water use efficiency (WUE) observed in response to nitrogen (N) fertilization were attributable to foliar N effects on carbon fixation rates or on stomatal control of water loss. Photosynthesis and transpiration were measured at different light intensities and ambient CO(2) molar fractions and comparisons were made between current-year shoots with average foliar N concentrations of 1.58% (High-N) and 1.25% (Low-N). Photosynthetic rates and foliar N concentrations were positively correlated. In response to light, photosynthesis and stomatal conductance were closely coupled and a similar coupling was observed in response to different ambient CO(2) concentrations. Partitioning the photosynthetic responses into mesophyll and stomatal components indicated that foliar N altered mesophyll conductance but not stomatal control of water loss. High-N shoots had significantly greater rates of photosynthesis and transpiration than Low-N shoots and, as a result, instantaneous WUE did not differ significantly between High-N and Low-N shoots.

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Leaf growth, photosynthesis and tissue water relations of greenhouse-grown Eucalyptus marginata seedlings in response to water deficits.

Leaf growth, rate of leaf photosynthesis and tissue water relations of shoots of Eucalyptus marginata Donn ex Sm. (jarrah) seedlings were studied during a soil drying and rewatering cycle in a greenhouse experiment. Rates of leaf growth and photosynthesis were sensitive to water deficits. The rate of leaf growth decreased linearly with predawn leaf water potential to reach zero at -1.5 MPa. Rate of leaf growth did not recover completely within the first three days after rewatering. Midday photosynthetic rates declined to 40% of those of well-watered seedlings at a predawn leaf water potential of -1.0 MPa and reached zero at -2.2 MPa. Photosynthetic rate recovered rapidly following rewatering and almost fully recovered by the second day after rewatering. All tissue water relations parameters, except the bulk modulus of elasticity, changed significantly as the soil dried and recovered completely by the third day after rewatering. Changes in osmotic pressure at full turgor of 0.4 MPa indicated considerable capacity for osmotic adjustment. However, because there was little osmotic adjustment until predawn leaf water potential fell below -1.5 MPa, this capacity would not have enhanced seedling growth, although it may have increased seedling survival. The sensitivity of photosynthesis and relative water content to water deficits suggests that greenhouse-grown E. marginata seedlings behave like mesophytic plants, even though E. marginata seedlings naturally grow in a drought-prone environment.

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An analytical solution for coupled leaf photosynthesis and stomatal conductance models.

Iterative solutions of coupled leaf photosynthesis and stomatal conductance equations sometimes yield bifurcated or chaotic solutions. An analytical solution for coupled leaf photosynthesis-stomatal conductance equations is preferred because an analytical model has specific and known roots, and partial derivatives can be taken to perform sensitivity analyses. I present an analytical solution for coupled leaf photosynthesis and stomatal conductance equations that are based on established biochemical and physiological theory.

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Recovery of photosynthesis in 1-year-old needles of unfertilized and fertilized Norway spruce (Picea abies (L.) Karst.) during spring.

Photosynthetic O(2) evolution and chlorophyll a fluorescence were measured in 1-year-old needles of unfertilized and fertilized trees of Norway spruce (Picea abies (L.) Karst.) during recovery of photosynthesis from winter inhibition in northern Sweden. Measurements were made under laboratory conditions at 20 degrees C. In general, the CO(2)-saturated rate of O(2) evolution was higher in needles of fertilized trees than in needles of unfertilized trees over a wide range of incident photon flux densities. Furthermore, the maximum photochemical efficiency of photosystem (PS) II, as indicated by the ratio of variable to maximum fluorescence (F(V)/F(M)) was higher in needles of fertilized trees than in needles of unfertilized trees. The largest differences in F(V)/F(M) between the two treatments occurred before the main recovery of photosynthesis from winter inhibition in late May. The rate of O(2) evolution was higher in needles of north-facing branches than in needles of south-facing branches in the middle of May. Simultaneous measurements of O(2) exchange and chlorophyll fluorescence indicated that differences in the rate of O(2) evolution between the two treatments were paralleled by differences in the rate of PS II electron transport determined by chlorophyll fluorescence. We suggest that, during recovery of photosynthesis from winter inhibition, the balance between carbon assimilation and PS II electron transport was maintained largely by adjustments in the nonphotochemical dissipation of excitation energy within PS II.

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Changes in rates of photosynthesis and respiration during needle development of loblolly pine.

Net photosynthetic rates of developing foliage and one-year-old foliage of loblolly pine (Pinus taeda L.) were measured under field conditions. In the subsequent year, net photosynthesis and dark respiration rates of current-year and one-year-old foliage were measured under controlled environmental conditions. Loblolly pine foliage grows slowly, reaching its final size 3.5 to 4 months after bud burst. Positive rates of net photosynthesis were recorded when the foliage was 13 and 18% of final length, in the controlled-environment and field study, respectively. However, because of high rates of dark respiration during the initial growth period, a positive diurnal carbon balance did not occur until foliage was about a third of final length (40 days after bud burst). Two months after bud burst, when foliage was about 55% of final length, its photosynthetic capacity exceeded that of one-year-old foliage. The highest rates of net photosynthesis were achieved when foliage was more than 90% fully expanded.

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Acclimation of photosynthesis and respiration to simulated climatic warming in northern and southern populations of Acer saccharum: laboratory and field evidence.

Physiological acclimation and genotypic adaptation to prevailing temperatures may influence forest responses to future climatic warming. We examined photosynthetic and respiratory responses of sugar maple (Acer saccharum Marsh.) from two portions of the species' range for evidence of both phenomena in a laboratory study with seedlings. A field study was also conducted to assess the impacts of temperature acclimation on saplings subjected to an imposed temperature manipulation (4 degrees C above ambient temperature). The two seedling populations exhibited more evidence of physiological acclimation to warming than of ecotypic adaptation, although respiration was less sensitive to short-term warming in the southern population than in the northern population. In both seedling populations, thermal compensation increased photosynthesis by 14% and decreased respiration by 10% in the warm-acclimated groups. Saplings growing in open-top field chambers at ambient temperature and 4 degrees C above ambient temperature showed evidence of temperature acclimation, but photosynthesis did not increase in response to the 4 degrees C warming. On the contrary, photosynthetic rates measured at the prevailing chamber temperature throughout three growing seasons were similar, or lower (12% lower on average) in saplings maintained at 4 degrees C above ambient temperature compared with saplings maintained at ambient temperature. However, the long-term photosynthetic temperature optimum for saplings in the field experiment was higher than it was for seedlings in either the 27 or the 31 degrees C growth chamber. Respiratory acclimation was also evident in the saplings in the field chambers. Saplings had similar rates of respiration in both temperature treatments, and respiration showed little dependence on prevailing temperature during the growing season. We conclude that photosynthesis and respiration in sugar maple have the potential for physiological acclimation to temperature, but exhibit a low degree of genetic adaptation. Some of the potential for acclimation to a 4 degrees C increase above a background of naturally fluctuating temperatures may be offset by differences in water relations, and, in the long term, may be obscured by the inherent variability in rates under field conditions. Nevertheless, physiologically based models should incorporate seasonal acclimation to temperature and permit ecotypic differences to influence model outcomes for those species with high genetic differentiation between regions.

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