Simultaneous measurement of oscillations in oxygen evolution and chlorophyll a fluorescence in leaf pieces

In spinach (Spinacia oleracea) and barley (Hordeum vulgare) leaves, chlorophyll a fluorescence and O₂ evolution have been measured simultaneously following re-illumination after a dark interval or when steady state photosynthesis has been perturbed by changes in the gas phase. In high CO₂ concentrations, both O₂ and fluorescence can display marked dampening oscillations that are antiparallel but slightly out of phase (a rise or fall in fluorescence anticipating a corresponding fall or rise in O₂ by about 10 to 15 seconds). Infrared gas analysis measurements showed that CO₂ uptake behaved like O₂ evolution both in the period of oscillation (about 1 minute) and in its relation to fluorescence. In the steady state, oscillations were initiated by increases in CO₂ or by increases or decreases in O₂. Oscillations in O₂ or CO₂ did not occur without associated oscillations in fluorescence and the latter were a sensitive indicator of the former. The relationship between such oscillations in photosynthetic carbon assimilation and chlorophyl a fluorescence is discussed in the context of the effect of ATP or NADPH consumption on known quenching mechanisms.     

Two-tier vessel for photoautotrophic high-density cultures

Two-tier vessels, developed for culturing of microalgae and cyanobacteria at high cell density on a shaken platform, were assembled from a flat lower chamber to be filled with a CO₂ buffer and an upper flat sterile chamber for the culture that was separated from the lower chamber by a porous polypropylene membrane. Diffusive gas exchange with the atmosphere was controlled by the O₂ outlet channel. Referred to surface area, rates of CO₂ transfer to a shaken weakly alkaline buffer solution across the membrane were higher than those reached on the conventional pathway through the free upper liquid surface. Membrane-mediated CO₂ supply enabled rapid growth of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 up to ultrahigh cell density. The biomass (dry weight) concentration of Synechococcus cultures reached more than 30 g L^?1 on a buffered medium with adequate concentrations of mineral nutrients. An increase of 15 to 20 g L^?1 was observed during repeated two-day cycles. Separate pathways for CO₂ supply and oxygen outlet prevented significant loss of CO₂. Convective gas flow through the oxygen outlet channel enabled the estimation of the O₂ generation rate. The permeability of the channel for diffusive O₂/N₂ exchange limited the O₂ concentration to a moderate value. It is concluded that shaken flat cultures using CO₂ supply through a porous hydrophobic membrane and diffusive release of O₂ through a separate pathway are promising for research on microalgae and cyanobacteria.     

A method for measuring the transfer of fixed nitrogen from free-living bacteria to higher plants using 15N2

An apparatus is described in which the root systems of up to ten plants can be simultaneously incubated with ¹⁵N₂ gas mixtures, while the aerial environment remains undisturbed. The size of incubation chamber and the number of chambers can be adjusted depending on the size and growth stage of the plants being tested. The apparatus has the facility for the recovery of gas mixtures, sampling and adjustment of gas concentrations, and control of soil moisture status within a sealed system. Possible experimental applications are suggested and some preliminary results presented which demonstrate bacterial nitrogen fixation and uptake into Sorghum seedlings.     

A method for equilibration chamber sampling and gas chromatographic analysis of the soil atmosphere

The method includes sampling of gases from an equilibration chamber permanently installed in the soil, transferring the sample to laboratory and subsequent measurement by gas chromatography. The equilibration chamber allows sampling of the gas phase both above and below the groundwater level, which is a major advantage. After significant concentration changes in non-saturated soils, gases in chambers regain equilibrium with the surrounding soils within 1–2 days. In the most unfavourable equilibration situations,i.e. in mineral subsoils with stagnant groundwater and very low biological activity, 90% equilibrium is attained in about 15 days. N₂, O₂+Ar, CO₂, CH₄, N₂O, H₂ and Ne, are measured on a series/bypass multi-column system, followed by a thermal conductivity detector.     

Resistance of citrus fruit to mass transport of water vapor and other gases

The resistance of oranges (Citrus sinensis L. Osbeck) and grapefruit (Citrus paradisi Macf.) to ethylene, O₂, CO₂, and H₂O mass transport was investigated anatomically with scanning electron microscope and physiologically by gas exchange measurements at steady state. The resistance of untreated fruit to water vapor is far less than to ethylene, CO₂ and O₂. Waxing partially or completely plugs stomatal pores and forms an intermittent cracked layer over the surface of fruit, restricting transport of ethylene, O₂, and CO₂, but not of water; whereas individual sealing of fruit with high density polyethylene films reduces water transport by 90% without substantially inhibiting gas exchange.

Stomata of harvested citrus fruits are essentially closed. However, ethylene, O₂ and CO₂ still diffuse mainly through the residual stomatal opening where the relative transport resistance (approximately 6,000 seconds per centimeter) depends on the relative diffusivity of each gas in air. Water moves preferentially by a different pathway, probably through a liquid aqueous phase in the cuticle where water conductance is 60-fold greater. Other gases are constrained from using this pathway because their diffusivity in liquid water is 10⁴-fold less than in air.

     

Chlorophyll fluorescence induction in anaerobic Scenedesmus obliquus

Fluorescence time curves (Kautsky effect) were studied in anaerobic Scenedesmus obliquus, with an apparatus capable of simultaneous recording of O₂ exchange, and far-red actinic illumination. Results, as interpreted in terms of electron transport reactions, suggest: In the course of becoming anaerobic, fluorescence induction undergoes a series of changes, indicating at least three different effects of the absence of O₂ on electron transport. (1) Immediately on removal of O₂, once the pool of intermediates between the two photo-systems is reduced by light reaction II, electron flow stops, resulting in high fluorescence yield and a cessation of O₂ evolution. O₂ appears to regulate linear electron flow and cyclic feedback of electrons to the intermediate pool. (2) An endogenous reductant formed anaerobically reduces the System II acceptors in the dark. The time course of this reduction is at least biphasic, indicative of inhomogeneity of the primary acceptor pool. Prolonged dark anaerobic treatment induces maximal initial fluorescence which decays rapidly in light and with a System I action spectrum. (3) Anaerobic treatment eventually results in deactivation of the oxidizing side of System II, limiting System II even when the acceptors are oxidized by System I pre-illumination.     

Continuous Measurements of the Free Dissolved CO(2) Concentration during Photosynthesis of Marine Plants: Evidence for HCO(3) Use in Chondrus crispus

An experimental system consisting of a gas exchange column linked to an assimilation chamber has been developed to record continuously the free dissolved CO₂ concentration in seawater containing marine plants. From experiments performed on the red macroalga Chondrus crispus (Rhodophyta, Gigartinales), this measurement is in agreement with the free CO₂ concentration calculated from the resistance to CO₂ exchanges in a biphasic system (gas and liquid) as earlier reported. The response time of this apparatus is short enough to detect, in conditions of constant pH, a photosynthesis-caused gradient between free CO₂ and HCO₃⁻ pools which half-equilibrates in 25 seconds. Abolished by carbonic anhydrase, the magnitude of this gradient increases with decreasing time of seawater transit from the chamber to the column apparatus. But its maximum magnitude (0.35 micromolar CO₂) is negligible compared to the difference between air and free CO₂ (11.4 micromolar CO₂). This illustrates the extent of the physical limiting-step occurring at the air-water interface when inorganic carbon consumption in seawater is balanced by dissolving gaseous CO₂. The direction of this small free CO₂/HCO₃⁻ gradient indicates that HCO₃⁻ is consumed during photosynthesis.     

Photoprotective function of photorespiration in Reaumuria soongorica during different levels of drought stress in natural high irradiance

Diurnal patterns of gas exchange and chlorophyll (Chl) fluorescence parameters of photosystem 2 (PS2) as well as H₂O₂ content were analyzed in Reaumuria soongorica (Pall.) Maxim., a perennial semi-shrub. The rate of photorespiration was estimated by combined measurement of gas exchange and Chl fluorescence. The rate of photorespiration increased with the increasing drought stress (DS). The ratio of carboxylation electron flow to oxygenation electron flow (J_c/J_o) and the maximal photochemical efficiency of PS2 (variable to maximum fluorescence ratio, F_v/F_m) decreased with the increasing DS. F_v/F_m in isonicotinic acid hydrazide (INH)-sprayed plants was lower than that in normal plants under moderate DS, but no significant difference was observed under severe DS. H₂O₂ content in INH-sprayed plants was significantly lower than that in normal plants under severe DS. Taken together, photorespiration in R. soongorica consumed excess electrons and protected photosynthetic apparatus under moderate DS, whereas it accelerated H₂O₂ accumulation markedly and induced the leaf abscission under severe DS.     

Effects of O(2) and CO(2) Concentrations on Quantum Yields of Photosystems I and II in Tobacco Leaf Tissue

The interactive effects of irradiance and O₂ and CO₂ levels on the quantum yields of photosystems I and II have been studied under steady-state conditions at 25°C in leaf tissue of tobacco (Nicotiana tabacum). Assessment of radiant energy utilization in photosystem II was based on changes in chlorophyll fluorescence yield excited by a weak measuring beam of modulated red light. Independent estimates of photosystem I quantum yield were based on the light-dark in vivo absorbance change at 830 nanometers, the absorption band of P700⁺. Normal (i.e. 20.5%, v/v) levels of O₂ generally enhanced photosystem II quantum yield relative to that measured under 1.6% O₂ as the irradiance approached saturation. Photorespiration is suspected to mediate such positive effects of O₂ through increases in the availability of CO₂ and recycling of orthophosphate. Conversely, at low intercellular CO₂ concentrations, 41.2% O₂ was associated with lower photosystem II quantum yield compared with that observed at 20.5% O₂. Inhibitory effects of 41.2% O₂ may occur in response to negative feedback on photosystem II arising from a build-up in the thylakoid proton gradient during electron transport to O₂. Covariation between quantum yields of photosystems I and II was not affected by concentrations of either O₂ or CO₂. The dependence of quantum yield of electron transport to CO₂ measured by gas exchange upon photosystem II quantum yield as determined by fluorescence was unaffected by CO₂ concentration.     

Radiorespirometer for continuous monitoring of chick embryo development.

A radiorespirometer is described that is capable of continuous monitoring of O₂ utilization, CO₂ and/or ¹⁴CO₂ production per minute, heart rate, and body activity of an embryonated egg while it develops for several days in a closed chamber with near normal pO₂ or other desired pO₂. Oxygen is supplied to the embryo by an electrolytic cell, and CO₂ is removed by a KOH solution flowing through a diffusion cell separated from the embryo chamber by a CO₂ permeable rubber membrane. The instrument permits selecting embryos for viability and developmental stage, according to their O₂ utilization per minute and respiratory quotient, without breaking the eggshell. Inoculation of the embryonated egg with ¹⁴C-labeled substrates, drugs, or toxins can occur without interfering with continuous recording of metabolic activity.     

Systems utilizing oxygenated leghemoglobin and myoglobin as sources of free dissolved O2 at low concentrations for experiments with bacteria

Oxygenated soybean leghemoglobin and sperm whale myoglobin have been used as sources of O₂ for respiring bacteria in experiments with no gas phase. These O₂-carrying hemoproteins provide dispersed free O₂ at concentrations defined by the kinetic constants of their oxygenation and deoxygenation, and their optical absorption spectra indicate the average concentration of free O₂. They can thus be used to study relationships between concentrations of free O₂, rates of respiration, and other metabolic processes in bacteria suspended in solutions of them. Three types of apparatus are described and examples of studies of respiration and nitrogen fixation by Rhizobium spp. are given.     

Plastocyanin as the possible site of photosynthetic electron transport inhibition by glutaraldehyde

Treatment of spinach chloroplasts with glutaraldehyde causes an inhibition in the electron transport chain between the two photosystems. Measurements of O₂ flash yields, pH exchange, and fluorescence induction show that the O₂ evolving apparatus, photosystem II and its electron acceptor pool are not affected. The behavior of P700 indicates that its reduction but not its oxidation, is severely inhibited. Cytochrome f is still reducible by photosystem II but also slowly oxidizable by photosystem I. The sensitivity of isolated plastocyanin to glutaraldehyde further supports the conclusion that glutaraldehyde inhibits at the plastocyanin level and thereby induces a break between P700 and cytochrome f.     

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins^1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N₂; <2 ppm O₂) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.     

The Rate of Photorespiration during Photosynthesis and the Relationship of the Substrate of Light Respiration to the Products of Photosynthesis in Sunflower Leaves

Single attached leaves of sunflower (Helianthus annus L. “Mennonite”) were supplied ¹⁴CO₂ of constant specific radioactivity in gas mixtures containing various CO₂ and O₂ concentrations. The ¹⁴CO₂ and CO₂ fluxes were measured concurrently in an open system using an ionization chamber and infrared gas analyzer.     

An Anaerobic Chemostat That Permits the Collection and Measurement of Fermentation Gases

A chemostat was designed to allow anaerobic growth in the culture vessel in the absence of a continuous stream of O₂-free gas. Produced gases were collected within the culture and collection vessels, and pressure build-up was prevented by allowing gases to expand into a collapsed football bladder. The culture overflow was collected in a flask, held at 0 C, that was emptied by applying a positive CO₂ pressure to the system. Ruminococcus albus, a H₂ and CO₂-producing anaerobe, was used to test the operation of the apparatus. H₂ production was measured by sampling the various gas spaces of known volume and measuring H₂ concentration by gas chromatography. Measurement of accumulated fermentation gases and the effects of the accumulation on fermentations can be studied with the apparatus.     

Ventilation of the giant nests of Atta leaf-cutting ants: does underground circulating air enter the fungus chambers?

Nest ventilation should be particularly relevant for the huge colonies of leaf-cutting ants, genus Atta. Considerable amounts of O₂ are consumed and CO₂ produced by both the fungus gardens and the ants inside nest chambers, which are located at deep soil layers characterized by high CO₂ and low O₂ concentrations. In this work, passive nest ventilation was investigated in field Atta capiguara and Atta laevigata nests, first, by evaluating air movements through the nest using propane as tracer gas as well as the CO₂ and O₂ concentrations of the circulating air, and second, by exposing the internal nest morphology with the use of cement casts and excavations. Results showed that even though outflow of CO₂-rich air and inflow of O₂-rich air occurred at high-placed and low-placed openings, respectively, supporting a wind-induced interpretation of air movements through the nest, circulating air was never detected inside fungus chambers. The CO₂ and O₂ levels inside the fungus chambers increased and decreased with increasing soil depth, respectively, and were in the range observed in the soil phase. Based on the underground nest architecture, it is concluded that although the external shape of the nest induces underground air circulation, the inflowing air is unable to directly reach the fungus chambers. It is argued that colony respiration completely depends on diffusive flows between the chamber air and the adjacent nest and soil atmospheres. Circulating air, although not directly renewing the air inside the nest chambers, may contribute to colony respiration by increasing the capacity of the nest and soil airs to act as an O₂-source and a CO₂-sink, because of the decrease in the CO₂ and the increase in the O₂ levels in the underground air phase. Possible adaptations of both ants and fungus to the high CO₂ and low O₂ concentrations usually found in soils are discussed.     

A sensitive continuous and discontinuous photometric determination of oxygen, carbon dioxide, and carbon monoxide in gases and fluids.

The paper describes a sensitive, rapid, and precise photometric method for the continuous and discontinuous determination of O₂, CO₂, and CO. The method is based on highly specific color reactions: O₂ is determined by its reaction with alkaline catechol + Fe²⁺ yielding intensively colored products, CO₂ is determined by its color reaction with a solution of fuchsin + hydrazine; and CO is determined by its reaction with hemoglobin. The basic experimental equipment is that of the AutoAnalyzer (cf.Wolf, Zander, and Lang, 1976, Anal. Biochem.74, 585), with an additional chamber for the injection of small gas samples in the case of the discontinuous analysis. Continuously analyzing in a standardized gas flow of 1 ml · min^?1 (STPD), the lower limits of the sensitivities are 50 ppm for O₂, 100 ppm for CO₂, and 50 ppm for CO. The discontinuous analysis of the three gases requires the basic experimental equipment plus an airtight chamber. The lower limits of the amounts are 0.1 μl (STPD) for O₂, 0.2 μl for CO₂, and 0.1 μl for CO.     

Influence of oxidative stressors on the photosynthetic apparatus of the methyl viologen-resistant mutant Prq20 of cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Delayed Chl a fluorescence and the CO₂-dependent O₂ exchange were measured to assess the effect of oxidative stress inducers methyl viologen and benzyl viologen, cumene hydroperoxide, menadione, and H₂O₂ as well as high irradiance on the photosynthetic apparatus of Synechocystis sp. PCC 6803 wild type and its methyl viologen-resistant mutant Prq20 with impaired regulatory gene prqR. The extent of damage upon exposure to viologens proved much smaller in the mutant; the causes of this are analyzed.     

PO2 of the metathoracic ganglion in response to progressive hypoxia in an insect

Mammals regulate their brain tissue P_O2 tightly, and only small changes in brain P_O2 are required to elicit compensatory ventilation. However, unlike the flow-through cardiovascular system of vertebrates, insect tissues exchange gases through blind-ended tracheoles, which may involve a more prominent role for diffusive gas exchange. We tested the effect of progressive hypoxia on ventilation and the P_O2 of the metathoracic ganglion (neural site of control of ventilation) using microelectrodes in the American locust, Schistocerca americana. In normal air (21 kPa), P_O2 of the metathoracic ganglion was 12 kPa. The P_O2 of the ganglion dropped as air P_O2 dropped, with ventilatory responses occurring when ganglion P_O2 reached 3 kPa. Unlike vertebrates, insects tolerate relatively high resting tissue P_O2 levels and allow tissue P_O2 to drop during hypoxia, activity and discontinuous gas exchange before activating convective or spiracular gas exchange. Tracheated animals, and possibly pancrustaceans in general, seem likely to generally experience wide spatial and temporal variation in tissue P_O2 compared with vertebrates, with important implications for physiological function and the evolution of oxygen-using proteins.