Analysis of the slow phases of the in vivo chlorophyll fluorescence induction curve. Changes in the redox state of Photosystem II electron acceptors and fluorescence emission from Photosystems I and II

An analysis of the photo-induced decline in the in vivo chlorophyll a fluorescence emission (Kautsky phenomenon) from the bean leaf is presented. The redox state of PS II electron acceptors and the fluorescence emission from PS I and PS II were monitored during quenching of fluorescence from the maximum level at P to the steady state level at T. Simultaneous measurement of the kinetics of fluorescence emission associated with PS I and PS II indicated that the ratio of PS I/PS II emission changed in an antiparallel fashion to PS II emission throughout the induction curve. Estimation of the redox state of PS II electron acceptors at given points during P to T quenching was made by exposing the leaf to additional excitation irradiation and determining the amount of variable PS II fluorescence generated. An inverse relationship was found between the proportion of PS II electron acceptors in the oxidised state and PS II fluorescence emission. The interrelationships between the redox state of PS II electron acceptors and fluorescence emission from PS I and PS II remained similar when the shape of the induction curve from P to T was modified by increasing the excitation photon flux density. The contributions of photochemical and non-photochemical quenching to the in vivo fluorescence decline from P to T are discussed.     

A spontaneous point mutation in the Rubisco large subunit gene impairing holoenzyme assembly renders the IVβ plastome mutant of Oenothera extremely light‐ and chilling sensitive

A spontaneously occurring chloroplast genome (plastome) mutant of Oenothera , IVβ, was identified as a single point mutation in the Rubisco large subunit gene (G337 → C), leading to an V113L exchange, which topologically occurs at the interface of two adjacent large subunits (LSU). The minor sterical hindrance of dimer formation by this amino acid exchange strongly impairs holoenzyme assembly, leading to an accumulation of a processing precursor of the holoenzyme, the B‐complex, consisting of one LSU and 14 units of chaperonine 60 (cpn60). It is associated with very low holoenzyme concentrations in the mutant tissue, but does not affect the kinetic properties of the enzyme once assembled. When grown under moderate or low light, leaf tissue containing the plastome mutant showed decreased Chl contents and Chl a / b ratios, increased relative carotenoid contents and violaxanthin deepoxidation activity, but very low CO₂ fixation and O₂ evolution rates and was very sensitive to photoinhibition. The light dependence of chlorophyll fluorescence quenching components at low temperature resembled an extremely chilling sensitive Oenothera genotype as compared to the wild‐type. The IVβ mutant thus behaves similarly to the Rubisco SSU antisense plants analysed by Stitt and co‐workers (summarised by Stitt and Schulze 1994) and gives an example of the possible influence of plastome mutations on the sensitivity of the photosynthetic apparatus to excess light by modifying the capacity of the Calvin cycle.     

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.     

Does greater leaf‐level photosynthesis explain the larger solar energy conversion efficiency of Miscanthus relative to switchgrass?

C₄ perennial grasses are being considered for bioenergy because of their high productivity and low inputs. In side-by-side replicated trials, Miscanthus ( Miscanthus x giganteus ) has previously been found more than twice as productive as switchgrass ( Panicum virgatum ). The hypothesis that this difference is attributable to higher leaf photosynthetic rates was tested on established plots of switchgrass and Miscanthus in central Illinois with >3300 individual measurements on 20 dates across the 2005 and 2006 growing seasons. Seasonally integrated leaf-level photosynthesis was 33% higher in Miscanthus than switchgrass ( P  < 0.0001). This increase in carbon assimilation comes at the expense of additional transpiration since stomatal conductance was on average 25% higher in Miscanthus ( P  < 0.0001). Whole-chain electron transport rate, measured simultaneously by modulated chlorophyll fluorescence, was similarly 23% higher in Miscanthus ( P  < 0.0001). Efficiencies of light energy transduction into whole chain photosynthetic electron transport, leaf nitrogen use and leaf water use were all significantly higher in Miscanthus. These may all contribute to its higher photosynthetic rates, and in turn, productivity. Systematic measurement of photosynthesis over two complete growing seasons in the field provides a unique dataset explaining why the productivity of these two species differs and for validating mechanistic production models for these emerging bioenergy crops.     

Odorant-induced kinetics of Ca<Superscript>2+</Superscript>, NADH,and oxidized flavoproteins in frog olfactory mucosa

A study was made of the odorant-induced changes in the fluorescence of the Ca²⁺-chlortetracycline-membrane complex, NADH, and oxidized flavoproteins in the frog olfactory epithelium. Cineole and vanillin induce faster changes than camphor and pentanol. The different kinetics of NADH and membrane calcium evoked by these odorants are attributed to the heterogeneity of the molecular mechanisms involved in olfactory signal transduction. By contrast, ammonia and β-mercaptoethanol permeate the olfactory cells and without second messengers inhibit the mitochondrial respiratory chain and suppress the motility of olfactory cilia.     

A mechanism for multiphasic uptake of solutes in plants

A molecular mechanism is proposed for unitary multiphasic uptake in which the carrier has two binding sites for the substrate. The first site binds n-1 molecules of substrate, and then one additional substrate molecule can become bound at the second binding site. Only this last molecule is transported in the operation of the carrier molecule. In the free state, the carrier can be activated to successive states with increasing affinities for the substrate in the two binding sites. The mechanism is resolved for the steady state conditions, obtaining a simple uptake rate equation, which fits the experimental data. Methods for determining the parameters of the equation are presented. Evidence other than kinetics is discussed for the mechanism. The mechanism also provides a physiological interpretation for multiphasic uptake: the active transport mechanisms (energy-requiring mechanisms) are prevented from operating at high substrate concentrations, thus preventing a waste of energy by the cells.     

Analysis of dihydroethidium fluorescence for the detection of intracellular and extracellular superoxide produced by NADPH oxidase

All methods used for quantitation of superoxide have limitations when it comes to differentiating between extracellular and intracellular sites of superoxide production. In the present study, we monitored dihydroethidium (DHE)-derived fluorescence at 570 nm, which indicates hydroxyethidium derived from reaction with superoxide produced by human leukemia cells (HL-60) and microvascular endothelial cells (HMEC-1). Phorbol-12-myristate 13-acetate (PMA; 100 ng/ml) caused an increase in fluorescence and lucigenin chemiluminescence in HL-60, which was abolished by superoxide dismutase (SOD; 600 U/ml) indicating that DHE detects extracellular superoxide. Furthermore, both HL-60 cells and HMEC-1 generated a fluorescence signal in the presence of DHE under resting conditions, which was unaffected by SOD, but abolished by polyethylene glycosylated-SOD (PEG-SOD) (100 U/ml) and MnTmPyP (25 μM), indicating that DHE also detects superoxide produced intracellularly. In HMEC-1, silencing of either Nox2 or Nox4 components of NADPH oxidase with small interference RNA (siRNA) resulted in a significant reduction in superoxide detected by both DHE fluorescence (Nox2 siRNA; 71 ± 6% and Nox4 siRNA 83 ± 7% of control) and lucigenin chemiluminescence (Nox2; 54 ± 6% and Nox4 74 ± 4% of control). In conclusion, DHE-derived fluorescence at 570 nm is a convenient method for detection of intracellular and extracellular superoxide produced by phagocytic and vascular NADPH oxidase.     

Fluorescence microscopy of the endomembrane system of living plant cells

Abstract The fluorochrome Auramine O has been evaluated as a fluorescent probe for components of the endomembrane system of living plant cells. At 0.001% w/v the compound did not inhibit seedling growth or cytoplasmic streaming but stained the nuclear envelope, endoplasmic reticulum and Golgi apparatus. The three-dimensional, structural interrelationships of these organelles in living tissues could be resolved after minimal tissue preparation. The method is also a valuable control treatment for use in conjunction with electron microscope fixation procedures. It provides a rapid means of examining dynamic changes in the endomembrane system associated with cell development and differentiation and could have application in monitoring the effects of applied physiological or chemical stress.