Thomas D. Sharkey

Photosynthesis in intact leaves of C3 plants: Physics, physiology and rate limitations

The instantaneous rate of photosynthetic CO2 assimilation in C3 plants has generally been studied in model systems such as isolated chloroplasts and algae. From these studies and from theoretical analyses of gas exchange behavior it is now possible to study the biochemistry of photosynthesis in intact leaves using a combination of methods, most of which are nondestructive.The limitations to the rate of photosynthesis can be divided among three general classes: (1) the supply or utilization of CO2, (2) the supply or utilization of light, and (3) the supply or utilization of phosphate. The first limitation is most readily studied by determining how the CO2 assimilation rate varies with the partial pressure of CO2 inside the leaf. The second limitation can be studied by determining the quantum requirement of photosynthesis. The third limitation is most easily detected as a loss of O2 sensitivity of photosynthesis.Measurement of fluorescence from intact leaves can give additional information about the various limitations. These methods are all non-destructive and so can be observed repeatedly as the environment of a leaf is changed. In addition, leaves can be quick-frozen and metabolite concentrations then measured to give more information about the limitations to intact leaf photosynthesis rates.In this review the physics and biochemistry of photosynthesis in intact C3 leaves, and the interface between physiology and photosynthesis—triose phosphate utilization—are discussed.ZusammenfassungDie photosynthetische CO2-Aufnahme wurde im allgemeinen an C3-Pflanzen während Kurzzeitexperimenten an Modellsystemen wie isolierten Chloroplasten und Algen erforscht. Aufgrund dieser Studien und der theoretischen Analysen über das Verhalten des Gasaustausches ist es nun möglich die Biochemie auch an intakten Blättern eingehender zu untersuchen und zwar, indem mehrere Methoden miteinander verknüpft angewandt werden, ohne das Blatt dabei zu verletzen.Die Limitierung der Photosyntheserate kann in drei grosse Gruppen unterteilt werden: 1. Angebot oder Nutzung von CO2, 2. Angebot oder Nutzung von Licht, 3. Verfügbarkeit oder Nutzung von intermediären Phosphatpoolen. Die erst erwähnte Begrenzung der Photosyntheserate kann einfach dadurch untersucht werden, indem man ermittelt, inwieweit die Rate der CO2-Aufnahme mit dem CO2-Partialdruck im Blattinneren variiert; die zweite, indem man den Quantenbedarf bestimmt. Die dritte Beschränkung der Rate kann leicht durch den Verlust der Sauerstoffempfindlichkeit in der Photosynthese entdeckt werden.Zusätzliche Information über die Art der verschiedenen Begrenzungen kann durch Fluorenszenzmessungen an intakten Blättern gewonnen werden. Da all diese Methoden am ganzen Blatt ausgeführt werden können, ohne es dabei zu beschädigen, können Messungen unter jeweils veränderten Umweltsbedingungen am gleichen Blatt wiederholt durchgeführt werden. Darüber hinaus können die Blätter schliesslich mit der Gefrierstop-Methode geerntet werden, um die Konzentration von Metaboliten zu messen, und so weitere Informationen über die Ursache der Begrenzung der Photosyntheserate im Bioassay zusätzlich zu den Ergebnissen am intakten Blatt erhalten werden.In diesem Übersichtsartikel werden die physikalischen und biochemischen Aspekten der Photosynthese sowie das Zusammenspiel von Physiologie und Photosynthese—Triosephosphate-Nutzung—diskutiert.

Biogenic hydrocarbons in the atmospheric boundary layer: A review

Nonmethane hydrocarbons are ubiquitous trace atmospheric constituents yet they control the oxidation capacity of the atmosphere. Both anthropogenic and biogenic processes contribute to the release of hydrocarbons to the atmosphere. In this manuscript, the state of the science concerning biosynthesis, transport, and chemical transformation of hydrocarbons emitted by the terrestrial biosphere is reviewed. In particular, the focus is on isoprene, monoterpenes, and oxygen-ated hydrocarbons. The generated science during the last 10 years is reviewed to explain and quantify hydrocarbon emissions from vegetation and to discern impacts of biogenic hydrocarbons on local and regional atmospheric chemistry. Furthermore, the physiological and environmental processes controlling biosynthesis and production of hydrocarbon compounds are reported on. Many advances have been made on measurement and modeling approaches developed to quantify hydrocarbon emissions from leaves and forest ecosystems. A synthesis of the atmospheric chemistry of biogenic hydrocarbons and their role in the formation of oxidants and aerosols is presented. The integration of biogenic hydrocarbon kinetics and atmospheric physics into mathematical modeling systems is examined to assess the contribution of biogenic hydrocarbons to the formation of oxidants and aerosols, thereby allowing us to study their impacts on the earths climate system and to develop strategies to reduce oxidant precursors in affected regions.

Water stress, temperature, and light effects on the capacity for isoprene emission and photosynthesis of kudzu leaves

Kudzu (Pueraria lobata (Willd) Ohwi.) is a vine which forms large, monospecific stands in disturbed areas of the southeastern United States. Kudzu also emits isoprene, a hydrocarbon which can significantly affect atmospheric chemistry including reactions leading to tropospheric ozone. We have studied physiological aspects of isoprene emission from kudzu so the ecological consequences of isoprene emission can be better understood. We examined: (a) the development of isoprene emission as leaves developed, (b) the interaction between photon flux density and temperature effects on isoprene emission, (c) isoprene emission during and after water stress, and (d) the induction of isoprene emission from leaves grown at low temperature by water stress or elevated temperature. Isoprene emission under standard conditions of 1000 μmol photons·m-2·s-1 and 30°C developed only after the leaf had reached full expansion, and was not complete until up to two weeks past the point of full expansion of the leaf. The effect of temperature on isoprene emission was much greater than found for other species, with a 10°C increase in temperature causing a eight-fold increase in the rate of isoprene emission. Isoprene emission from kudzu was stimulated by increases in photon flux density up to 3000 μmol photons·m-2·s-1. In contrast, photosynthesis of kudzu was saturated at less than 1000 μmol·m-2·s-1 photon flux density and was reduced at high temperature, so that up to 20% of the carbon fixed in photosynthesis was reemitted as isoprene gas at 1000 μmol photons·m-2·s-1 and 35°C. Withholding water caused photosynthesis to decline nearly to zero after several days but had a much smaller effect on isoprene emission. Following the relief of water stress, photosynthesis recovered to the prestress level but isoprene emission increased to about five times the prestress rate. At 1000 μmol photons·m-2·s-1 and 35°C as much as 67% of the carbon fixed in photosynthesis was reemitted as isoprene eight days after water stress. Leaves grown at less than 20°C did not make isoprene until an inductive treatment was given. Inductive treatments included growth at 24°C, leaf temperature of 30°C for 5 h, or witholding water from plants. With the new information on temperature and water stress effects on isoprene emission, we speculate that isoprene emission may help plants cope with stressful conditions.

An improved model of C3 photosynthesis at high CO2: Reversed O2 sensitivity explained by lack of glycerate reentry into the chloroplast

Current models of C3 photosynthesis incorporate a phosphate limitation to carboxylation which arises when the capacity for starch and sucrose synthesis fails to match the capacity for the production of triose phosphates in the Calvin cycle. As a result, the release of inorganic phosphate in the chloroplast stroma fails to keep pace with its rate of sequestration into triose phosphate, and phosphate becomes limiting to photosynthesis. Such a model predicts that when phosphate is limiting, assimilation becomes insensitive to both CO2 and O2, and is thus incapable of explaining the experimental observation that assimilation, under phosphate-limited conditions, frequently exhibits reversed sensitivity to both CO2 and O2, i.e., increasing O2 stimulates assimilation and increasing CO2 inhibits assimilation. We propose a model which explains reversed sensitivity to CO2 and O2 by invoking the net release of phosphate in the photorespiratory oxidation cycle. In order for this to occur, some fraction of the glycollate carbon which leaves the stroma and which is recycled to the chloroplast by the photorespiratory pathway as glycerate must remain in the cytosol, perhaps in the form of amino acids. In that case, phosphate normally used in the stromal glycerate kinase reaction to generate PGA from glycerate is made available for photophosphorylation, stimulating RuBP regeneration and assimilation. The model is parameterized for data obtained on soybean and cotton, and model behavior in response to CO2, O2, and light is demonstrated.

A GAS-EXCHANGE STUDY OF PHOTOSYNTHESIS AND ISOPRENE EMISSION IN QUERCUS RUBRA L.

We have investigated the signals which affect the rate of isoprene emission from photosynthesizing leaves of red oak (Quercus rubra L.) using analytical gas-exchange techniques, chlorophyll-fluorescence measurements, and inhibitor feeding. Isoprene emission increased with increasing photon flux density at low CO2 but much less so at high CO2 partial pressure. Photosynthetic CO2 assimilation exhibited the opposite behavior. In CO2-free air, isoprene emission was reduced; above 500 μbar CO2 partial pressure, isoprene emission was also reduced. The high-CO2 effect appeared to be related to low ATP levels which can occur during feedback-limited photosynthesis. At high temperature, which can prevent feedback limitations, isoprene emission remained high as CO2 partial pressure was increased. After exposing the leaves to darkness, isoprene emission declined over 15 min, while photosynthesis stopped within 2 min. Adding far-red light to stimulate cyclic photo-phosphorylation during the post-illumination period stimulated isoprene emission. These analyses lead us to propose that the rate of isoprene emission is regulated by ATP. Analysis of transients indicated that isoprene emission is also related to photosynthetic carbon metabolism. Inhibitor feeding indicated that 3-phosphoglyceric acid and 1,3-bisphosphoglyceric acid are possible candidates for the link between photosynthetic carbon metabolism and the regulation of isoprene emission. Given the ATP dependence, we suggest that the concentration of 1,3-bisphosphoglyceric acid may exert control over the rate of isoprene emission from oak leaves.

Isoprene Increases Thermotolerance of Isoprene-Emitting Species

Isoprene-emitting plants lose a large portion of their assimilated C as isoprene. Because isoprene synthesis can be regulated, it has been assumed that isoprene benefits the plant. Since the rate of isoprene emission from leaves is highly responsive to temperature, we hypothesized that isoprene benefits plants by increasing their thermotolerance. We used three methods to measure isopreneinduced thermotolerance in leaves. Each technique assayed thermotolerance under conditions that suppressed endogenous isoprene synthesis. When measured by chlorophyll fluorescence, thermotolerance of kudzu (Pueraria lobata [Willd.] Ohwi.) leaves increased as much as 4[deg]C in very low light. With higher light, isoprene increased thermotolerance of kudzu leaves by as much as 10[deg]C. When measured as the temperature at which photosynthesis declined to zero, thermotolerance increased with added isoprene by 2.5[deg]C. All three measures of thermotolerance were dose dependent. Both fluorescence techniques also showed isoprene-induced thermotolerance in white oak (Quercus alba L.). Thermotolerance was not observed in bean (Phaseolus vulgaris var Linden), a species that does not emit isoprene. None of the experiments was designed to determine the mechanism of thermotolerance, but we theorize that isoprene functions by enhancing hydrophobic interactions in membranes.

Evolution of the Isoprene Biosynthetic Pathway in Kudzu

Isoprene synthase converts dimethylallyl diphosphate, derived from the methylerythritol 4-phosphate (MEP) pathway, to isoprene. Isoprene is made by some plants in substantial amounts, which affects atmospheric chemistry, while other plants make no isoprene. As part of our long-term study of isoprene synthesis, the genetics of the isoprene biosynthetic pathway of the isoprene emitter, kudzu (Pueraria montana), was compared with similar genes in Arabidopsis (Arabidopsis thaliana), which does not make isoprene. The MEP pathway genes in kudzu were similar to the corresponding Arabidopsis genes. Isoprene synthase genes of kudzu and aspen (Populus tremuloides) were cloned to compare their divergence with the divergence seen in MEP pathway genes. Phylogenetic analysis of the terpene synthase gene family indicated that isoprene synthases are either within the monoterpene synthase clade or sister to it. In Arabidopsis, the gene most similar to isoprene synthase is a myrcene/ocimene (acyclic monoterpenes) synthase. Two phenylalanine residues found exclusively in isoprene synthases make the active site smaller than other terpene synthase enzymes, possibly conferring specificity for the five-carbon substrate rather than precursors of the larger isoprenoids. Expression of the kudzu isoprene synthase gene in Arabidopsis caused Arabidopsis to emit isoprene, indicating that whether or not a plant emits isoprene depends on whether or not it has a terpene synthase capable of using dimethylallyl diphosphate.

Daylength and Circadian Effects on Starch Degradation and Maltose Metabolism

Transitory starch is stored during the day inside chloroplasts and broken down at night for export. Maltose is the primary form of carbon export from chloroplasts at night. We investigated the influence of daylength and circadian rhythms on starch degradation and maltose metabolism. Starch breakdown was faster in plants of Arabidopsis (Arabidopsis thaliana) ecotype Wassilewskija growing in long days. Transcript levels of genes encoding enzymes involved in starch degradation and maltose metabolism showed a strong diurnal rhythm. Under altered photoperiods, the transcript levels and the rate of starch degradation changed within one day/night cycle. However, the amount of proteins involved in starch degradation was maintained relatively constant throughout the day/night cycle. To investigate whether the diurnal cycling of the transcript levels is only a response to light or is also regulated by a circadian clock, we measured the amount of messenger RNAs in Arabidopsis leaves under continuous light and continuous darkness. The expression of genes encoding starch degradation-related enzymes was under very strong circadian control in continuous light. Under continuous light, the amount of maltose also showed a strong endogenous rhythm close to 24 h, indicating that maltose metabolism is under circadian control. Light is necessary for the cycling of transcript levels and maltose levels. Under continuous darkness, these genes were barely expressed, and no cycling of maltose levels was observed.

Light-emitting diodes as a light source for photosynthesis research

Light-emitting diodes (LED) can provide large fluxes of red photons and so could be used to make lightweight, efficient lighting systems for photosynthetic research. We compared photosynthesis, stomatal conductance and isoprene emission (a sensitive indicator of ATP status) from leaves of kudzu (Pueraria lobata (Willd) Ohwi.) enclosed in a leaf chamber illuminated by LEDs versus by a xenon arc lamp. Stomatal conductance was measured to determine if red LED light could sufficiently open stomata. The LEDs produced an even field of red light (peak emission 656±5 nm) over the range of 0–1500 μmol m-2 s-1. Under ambient CO2 the photosynthetic response to red light deviated slightly from the response measured in white light and stomatal conductance followed a similar pattern. Isoprene emission also increased with light similar to photosynthesis in white light and red light. The response of photosynthesis to CO2 was similar under the LED and xenon arc lamps at equal photosynthetic irradiance of 1000 μmol m-2 s-1. There was no statistical difference between the white light and red light measurements in high CO2. Some leaves exhibited feedback inhibition of photosynthesis which was equally evident under irradiation of either lamp type. Photosynthesis research including electron transport, carbon metabolism and trace gas emission studies should benefit greatly from the increased reliability, repeatability and portability of a photosynthesis lamp based on light-emitting diodes.

The relationship between steady-state gas exchange of bean leaves and the levels of carbon-reduction-cycle intermediates

The relationship between the gas-exchange characteristics of attached leaves of Phaseolus vulgaris L. and the pool sizes of several carbon-reduction-cycle intermediates was examined. After determining the rate of CO2 assimilation at known intercellular CO2 pressure, O2 pressure and light, the leaf was rapidly killed (<0.1 s) and the levels of ribulose-1,5-bisphosphate (RuBP), 3-phosphoglyceric acid (PGA), fructose-1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, glyceraldehyde-3-phosphate, and dihydroxyacetone phosphate were measured. In 210 mbar O2, photosynthesis appeared RuBP-saturated at low CO2 pressure and RuBP-limited at high CO2 pressure. In 21 mbar (2%) O2, the level of RuBP always appeared saturating. Very high levels of PGA and other phosphate-containing compounds were found with some conditions, especially under low oxygen.