Anomalous Extracellular Diffusion in Rat Cerebellum

Extracellular space (ECS) is a major channel transporting biologically active molecules and drugs in the brain. Diffusion-mediated transport of these substances is hindered by the ECS structure but the microscopic basis of this hindrance is not fully understood. One hypothesis proposes that the hindrance originates in large part from the presence of dead-space (DS) microdomains that can transiently retain diffusing molecules. Because previous theoretical and modeling work reported an initial period of anomalous diffusion in similar environments, we expected that brain regions densely populated by DS microdomains would exhibit anomalous extracellular diffusion. Specifically, we targeted granular layers (GL) of rat and turtle cerebella that are populated with large and geometrically complex glomeruli. The integrative optical imaging (IOI) method was employed to evaluate diffusion of fluorophore-labeled dextran (MW 3000) in GL, and the IOI data analysis was adapted to quantify the anomalous diffusion exponent d_w from the IOI records. Diffusion was significantly anomalous in rat GL, where d_w reached 4.8. In the geometrically simpler turtle GL, d_w was elevated but not robustly anomalous (d_w = 2.6). The experimental work was complemented by numerical Monte Carlo simulations of anomalous ECS diffusion in several three-dimensional tissue models containing glomeruli-like structures. It demonstrated that both the duration of transiently anomalous diffusion and the anomalous exponent depend on the size of model glomeruli and the degree of their wrapping. In conclusion, we have found anomalous extracellular diffusion in the GL of rat cerebellum. This finding lends support to the DS microdomain hypothesis. Transiently anomalous diffusion also has a profound effect on the spatiotemporal distribution of molecules released into the ECS, especially at diffusion distances on the order of a few cell diameters, speeding up short-range diffusion-mediated signals in less permeable structures.     

Translational Diffusion of Macromolecule-sized Solutes in Cytoplasm and Nucleus

Fluorescence recovery after photobleaching (FRAP) was used to quantify the translational diffusion of microinjected FITC-dextrans and Ficolls in the cytoplasm and nucleus of MDCK epithelial cells and Swiss 3T3 fibroblasts. Absolute diffusion coefficients (D) were measured using a microsecond-resolution FRAP apparatus and solution standards. In aqueous media (viscosity 1 cP), D for the FITC-dextrans decreased from 75 to 8.4 × 10⁻⁷ cm²/s with increasing dextran size (4–2,000 kD). D in cytoplasm relative to that in water (D/D_o) was 0.26 ± 0.01 (MDCK) and 0.27 ± 0.01 (fibroblasts), and independent of FITC-dextran and Ficoll size (gyration radii [R_G] 40–300 Å). The fraction of mobile FITC-dextran molecules (f_mob), determined by the extent of fluorescence recovery after spot photobleaching, was >0.75 for R_G < 200 Å, but decreased to <0.5 for R_G > 300 Å. The independence of D/D_o on FITC-dextran and Ficoll size does not support the concept of solute “sieving” (size-dependent diffusion) in cytoplasm. Photobleaching measurements using different spot diameters (1.5–4 μm) gave similar D/D_o, indicating that microcompartments, if present, are of submicron size. Measurements of D/D_o and f_mob in concentrated dextran solutions, as well as in swollen and shrunken cells, suggested that the low f_mob for very large macromolecules might be related to restrictions imposed by immobile obstacles (such as microcompartments) or to anomalous diffusion (such as percolation). In nucleus, D/D_o was 0.25 ± 0.02 (MDCK) and 0.27 ± 0.03 (fibroblasts), and independent of solute size (R_G 40–300 Å). Our results indicate relatively free and rapid diffusion of macromolecule-sized solutes up to approximately 500 kD in cytoplasm and nucleus.     

Diffusion of Exchangeable Water in Cortical Bone Studied by Nuclear Magnetic Resonance

The rate-limiting step in the delivery of nutrients to osteocytes and the removal of cellular waste products is likely diffusion. The transport of osteoid water across the mineralized matrix of bone was studied by proton nuclear magnetic resonance spectroscopy and imaging by measuring the diffusion fluxes of tissue water in cortical bone specimens from the midshaft of rabbit tibiae immersed in deuterium oxide. From the diffusion coefficient (D_a = (7.8 ± 1.5) × 10⁻⁷ cm²/s) measured at 40°C (close to physiological temperature), it can be inferred that diffusive transport of small molecules from the bone vascular system to the osteocytes occurs within minutes. The activation energy for water diffusion, calculated from D_a measured at four different temperatures, suggests that the interactions between water molecules and matrix pores present significant energy barriers to diffusion. The spatially resolved profile of D_a perpendicular to the cortical surface of the tibia, obtained using a finite difference model, indicates that diffusion rates are higher close to the endosteal and periosteal surfaces, decreasing toward the center of the cortex. Finally, the data reveal a water component (∼30%) diffusing four orders of magnitude more slowly, which is ascribed to water tightly bound to the organic matrix and mineral phase.     

Transient state isoelectric focusing. Experimental determination of apparent diffusion coefficients in polyacrylamide gels

A photoelectric scanning assembly utilizing uv absorption optics and an on-line digital data acquisition and processing system has been used to follow kinetically zone spreading during the defocusing stage (absence of electric field) of transient state isoelectric focusing (TRANSIF) in polyacrylamide gels. Measurement of the variance (σ²) of a diffusing zone as a function of time yields a linear relationship, the slope of which corresponds to the apparent diffusion coefficient (D) of the protein. A linear relationship is also obtained when the logarithm of the apparent diffusion coefficients (logD) are plotted vs acrylamide concentration (T). This relationship can be used to extrapolate D to zero gel concentration. The apparent diffusion coefficient measured in this way is significantly larger than the true diffusion coefficient. The slope of the plot logD vs T, designated C_R, is expected to be a measure of molecular size related to the retardation coefficient in polyacrylamide gel electrophoresis.     

Astrocytes and extracellular matrix in extrasynaptic volume transmission

Volume transmission is a form of intercellular communication that does not require synapses; it is based on the diffusion of neuroactive substances across the brain extracellular space (ECS) and their binding to extrasynaptic high-affinity receptors on neurons or glia. Extracellular diffusion is restricted by the limited volume of the ECS, which is described by the ECS volume fraction α, and the presence of diffusion barriers, reflected by tortuosity λ, that are created, for example, by fine astrocytic processes or extracellular matrix (ECM) molecules. Organized astrocytic processes, ECM scaffolds or myelin sheets channel the extracellular diffusion so that it is facilitated in a certain direction, i.e. anisotropic. The diffusion properties of the ECS are profoundly influenced by various processes such as the swelling and morphological rebuilding of astrocytes during either transient or persisting physiological or pathological states, or the remodelling of the ECM in tumorous or epileptogenic tissue, during Alzheimer''s disease, after enzymatic treatment or in transgenic animals. The changing diffusion properties of the ECM influence neuron–glia interaction, learning abilities, the extent of neuronal damage and even cell migration. From a clinical point of view, diffusion parameter changes occurring during pathological states could be important for diagnosis, drug delivery and treatment.     

Long-range nonanomalous diffusion of quantum dot-labeled aquaporin-1 water channels in the cell plasma membrane

Aquaporin-1 (AQP1) is an integral membrane protein that facilitates osmotic water transport across cell plasma membranes in epithelia and endothelia. AQP1 has no known specific interactions with cytoplasmic or membrane proteins, but its recovery in a detergent-insoluble membrane fraction has suggested possible raft association. We tracked the membrane diffusion of AQP1 molecules labeled with quantum dots at an engineered external epitope at frame rates up to 91 Hz and over times up to 6 min. In transfected COS-7 cells, >75% of AQP1 molecules diffused freely over ∼7 μm in 5 min, with diffusion coefficient, D_1-3 ∼ 9 × 10⁻¹⁰ cm²/s. In MDCK cells, ∼60% of AQP1 diffused freely, with D_1-3 ∼ 3 × 10⁻¹⁰ cm²/s. The determinants of AQP1 diffusion were investigated by measurements of AQP1 diffusion following skeletal disruption (latrunculin B), lipid/raft perturbations (cyclodextrin and sphingomyelinase), and bleb formation. We found that cytoskeletal disruption had no effect on AQP1 diffusion in the plasma membrane, but that diffusion was increased greater than fourfold in protein de-enriched blebs. Cholesterol depletion in MDCK cells greatly restricted AQP1 diffusion, consistent with the formation of a network of solid-like barriers in the membrane. These results establish the nature and determinants of AQP1 diffusion in cell plasma membranes and demonstrate long-range nonanomalous diffusion of AQP1, challenging the prevailing view of universally anomalous diffusion of integral membrane proteins, and providing evidence against the accumulation of AQP1 in lipid rafts.     

Contribution of dead-space microdomains to tortuosity of brain extracellular space

The extracellular space (ECS) of the brain is a major channel for intercellular communication, nutrient and metabolite trafficking, and drug delivery. The dominant transport mechanism is diffusion, which is governed by two structural parameters, tortuosity and volume fraction. Tortuosity (lambda) represents the hindrance imposed on the diffusing molecules by the tissue in comparison with an obstacle-free medium, while volume fraction (alpha) is the proportion of tissue volume occupied by the ECS. Diffusion of small ECS markers can be exploited to measure lambda and alpha. In healthy brain tissue, lambda is about 1.6 but increases to 1.9-2.0 in pathologies that involve cellular swelling. Previously it was thought that lambda could be explained by the circumnavigation of diffusing molecules around cells. Numerical models of assemblies of convex cells, however, give an upper limit of about 1.23 for lambda. Therefore, additional factors must be responsible for lambda in brain. In principle, two mechanisms could account for the measured value: a more complex ECS geometry or an extracellular macromolecular matrix. Here we review recent work in ischemic tissue suggesting concave geometrical formations, dead-space microdomains, as a major determinant of extracellular tortuosity. A theoretical model of lambda based on diffusion dwell times supports this hypothesis and predicts that, in ischemia, dead spaces occupy approximately 60% of ECS volume fraction leaving only approximately 40% for well-connected channels. It is further proposed that dead spaces are present in healthy brain tissue where they constitute about 40% of alpha. The presence of dead-space microdomains in the ECS implies microscopic heterogeneity of extracellular channels with fundamental implications for molecular transport in brain.     

Effects of Charge on Osmotic Reflection Coefficients of Macromolecules in Fibrous Membranes

A model based on continuum hydrodynamics and electrostatics was developed to predict the combined effects of molecular charge and size on the osmotic reflection coefficient (σ_o) of a macromolecule in a fibrous membrane, such as a biological hydrogel. The macromolecule was represented as a sphere with a constant surface charge density, and the membrane was assumed to consist of an array of parallel fibers of like charge, also with a constant surface charge density. The flow was assumed to be parallel to the fiber axes. The effects of charge were included by computing the electrostatic free energy for a sphere interacting with an array of fibers. It was shown that this energy could be approximated using a pairwise additivity assumption. Results for σ_o were obtained for two types of negatively charged fibers, one with properties like those of glycosaminoglycan chains, and the other for thicker fibers having a range of charge densities. Using physiologically reasonable fiber spacings and charge densities, σ_o for bovine serum albumin in either type of fiber array was shown to be much larger than that for an uncharged system. Given the close correspondence between σ_o and the reflection coefficient for filtration, the results suggest that the negative charge of structures such as the endothelial surface glycocalyx is important in minimizing albumin loss from the circulation.     

The diffusion-solubility of oxygen in lipid bilayers

The product, D_oα, of the oxygen diffusion coefficient, D_o, and the oxygen solubility, α, is determined in phosphatidylcholine bilayers at temperatures above the lipid phase transitions from ESR spin-exchange measurements. The resulting values of D_oα are in good agreement with those obtained from fluorescence-quenching experiments. The use of fatty acid spin labels makes it possible to measure D_oα as a function of the coordinate perpendicular to the bilayer surface. The results indicate that D_oα is a strong function of this coordinate; it is greatest in the bilayer center and least near the bilayer head groups.     

Appendix: A model of plaque formation

Equations describing plaque formation in soft agar have been based on certain simplifying assumptions, for which data are presented. The derived equations permit one to calculate (i) average plaque size as a function of the initial density of indicator cells (D_o), (ii) the number of cells lysed per plaque as a function of D_o, and (iii) the cumulative number of cells lysed at various stages of plaque development. The calculated values agree well with those determined experimentally.     

Differential interferometry in the analytical ultracentrifuge. II. General applications

Various ways of applying differential interferometry to ultracentrifugal analyses are examined and several analytical techniques are established. In transport and moving boundary methods, the sedimentation coefficient is more precisely determined in the differential interference system than in the schlieren optical system because fringe measurement accuracy is much higher in the former system. Compared to interference and absorption optics, the differential interferometer provides a more exact s value in the transport method since an accurate calculation procedure can be adopted. Moreover, the following advantages of differential interferometry are noted. Determination of the initial solute concentration, which must be done in the usual interference method, is unnecessary in this sedimentation equilibrium method. Regardless of the partial loss of solute from the observed system due to rapid precipitation or adsorption to the cell wall during centrifugation, the molecular weight of the rest of the solute can be determined exactly. The diffusion coefficient can be determined accurately by fringe displacement analysis at the hinge point during the transient state. Together with the molecular weight and diffusion coefficient, the partial specific volume and sedimentation coefficient of a solute can be obtained from the result of a single low-speed centrifugation when the sample solutions in H₂O and D₂O are compared.     

Effect of host framework on the diffusion process in microporous material: a molecular dynamics simulation investigation

One of the central assumptions when a particle moves through a window in microporous materials is that interaction of the diffusing particle with the silicon (Si) and aluminum (Al) atoms of the framework can be neglected, as the presence of bulkier oxygen in the host structure is thought to hinder close proximity of the diffusing particle to Si and Al. We examine this assumption, exploring the diffusion path and cross-checking the bottleneck associated with the diffusion process. Our study reveals that short-range interactions between the diffusing species and Si/Al of the host have a significant effect on the diffusion process. Guest–host interaction energy increases significantly if interaction between Si and Al atoms with the diffusing species is considered. The self-diffusion coefficient (D) decreases significantly in the linear regime, whereas in the anomalous regime, surprisingly, D increases. The increase in D is due to a decrease in the activation energy in the anomalous regime, whereas in the linear regime, activation energy increases, thus D decreases.

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Graphical abstract a Interaction energies (E _a) for different LJ potential for guest–guest interactions (σ_gg) along the diffusion path; b correspondingdiffusivity values

     

Specific binding protein for 1,25-dihydroxyvitamin D3 in bovine mammary gland

Specific binding proteins for 1,25-dihydroxyvitamin D₃ were identified in bovine mammary tissue obtained from lactating and non-lactating mammary glands by sucrose density gradient centrifugation. The macromolecules had characteristic sedimentation coefficients of 3.5-3.7 S. The interaction of l,25-dihydroxy[³H]vitamin D₃ with the macromolecule of the mammary gland cytosol occurred at low concentrations, was saturable, and was a high affinity interaction (K_d = 4.2 × 10^?10M at 25 °C). Binding was reversed by excess unlabeled 1,25-dihydroxyvitamin D₃, was destroyed by heat and/or incubation with trypsin. It is thus inferred that this macromolecule is protein as it is not destroyed by ribonuclease or deoxyribonuclease. 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, and vitamin D₃ did not effectively compete with 1,25-dihydroxyvitamin D₃ for binding to cytosol of mammary tissue at near physiological concentrations of these analogs, thus demonstrating the specificity of the binding protein for 1,25-dihydroxyvitamin D₃. In vitro subcellular distribution of 1,25-dihydroxy[³H]vitamin D₃ demonstrated a time- and temperature-dependent movement of the hormone from the cytoplasm to the nucleus. By 90 min at 25 °C 72% of the 1,25-dihydroxy[³H]vitamin D₃ was associated with the nucleus. In addition a 5–6 S macromolecule which binds 25-hydroxy[³H]vitamin D₃ was demonstrated in mammary tissue. Finally, it is possible that the receptor-hormone complex present in mammary tissue may function in a manner analogous to intestinal tissue, resulting in the control of calcium transport by 1,25-dihydroxyvitamin D₃ in this tissue.     

High-Bandwidth Protein Analysis Using Solid-State Nanopores

High-bandwidth measurements of the ion current through hafnium oxide and silicon nitride nanopores allow the analysis of sub-30 kD protein molecules with unprecedented time resolution and detection efficiency. Measured capture rates suggest that at moderate transmembrane bias values, a substantial fraction of protein translocation events are detected. Our dwell-time resolution of 2.5 μs enables translocation time distributions to be fit to a first-passage time distribution derived from a 1D diffusion-drift model. The fits yield drift velocities that scale linearly with voltage, consistent with an electrophoretic process. Further, protein diffusion constants (D) are lower than the bulk diffusion constants (D₀) by a factor of ∼50, and are voltage-independent in the regime tested. We reason that deviations of D from D₀ are a result of confinement-driven pore/protein interactions, previously observed in porous systems. A straightforward Kramers model for this inhibited diffusion points to 9- to 12-kJ/mol interactions of the proteins with the nanopore. Reduction of μ and D are found to be material-dependent. Comparison of current-blockage levels of each protein yields volumetric information for the two proteins that is in good agreement with dynamic light scattering measurements. Finally, detection of a protein-protein complex is achieved.     

Repeated Electroconvulsive Shock Induces Changes in High-affinity [3H]-ouabain Binding to Rat Striatal Membranes

Repeated electroconvulsive shock is an effective treatment for affective disorders. Striatum, hippocampus and brainstem are involved in affective disorders. Sodium–potassium/ATPase is of paramount importance for the proper functioning of the brain and its involvement in the affective disorders has been claimed for a long time. Sodium–potassium/ATPase has an extracellular regulatory binding site to which cardiotonic glycosides, such as ouabain, bind to, thus regulating the activity of the enzyme. Endogenous “ouabain-like” substances exist in the brain and their actions on the sodium–potassium/ATPase resemble ouabain biological properties. The aim of this work was to determine if electroconvulsive shock (ECS) would induce changes in the high-affinity binding of ouabain to the sodium–potassium/ATPase from rat brain regions. Adult, male Wistar rats received one (ECS×1 group) or seven electroshocks (ECS×7 group) delivered daily through ear-clips electrodes. Control rats received the same manipulations; however, no current was delivered through the electrodes (SHAM×1 and SHAM×7 groups). All groups were sacrificed 24 h after the last ECS session. The B _max and K _D of high-affinity [³H]-ouabain binding were determined in crude membrane preparations from the striatum, hippocampus and brainstem. The results obtained showed a statistically significant increase in the affinity of [³H]-ouabain (lower K _D) to striatal membranes in those rats receiving seven ECS. In the striatum there was no change in the K _D after one ECS; as well as there was no change in the B _max after a single or seven ECS. High-affinity [³H]-ouabain binding to hippocampus and brainstem did not reveal any significant differences either in K _D or B _max after one or seven ECS. The increased affinity of ouabain to the striatal sodium–potassium/ATPase induced by repeated ECS suggests an increased interaction in vivo of the endogenous “ouabain-like” substances with the enzyme and the involvement of the extracellular regulatory allosteric ouabain binding site in the striatal sodium–potassium/ATPase in the effects of electroconvulsive shock.     

Role of Hydrogen-Bonding in Nonelectrolyte Diffusion through Dense Artificial Membranes

The diffusion of two series of alcohols and amides through complex cellulose acetate membranes was studied. The thin dense part of these membranes behaves as a nonporous layer of low water content. In this layer, called the skin, the solute diffusion coefficients, ω, depend upon size, steric configuration, and the partition coefficient, K₈, between membrane and bathing solution. From the experimental values of ω and K₈, the over-all friction, f, experienced by the solutes in the membrane was computed. It was found that f depends upon the chemical nature of the solute and is related to hydrogen-bonding ability. In the coarse, porous layer of the cellulose acetate membrane, diffusion occurs mainly through aqueous channels. In this instance also the hydrogen-bonding ability of the solute seems to exercise a smaller but significant influence.     

The Impact of Alpha-Syntrophin Deletion on the Changes in Tissue Structure and Extracellular Diffusion Associated with Cell Swelling under Physiological and Pathological Conditions

Aquaporin-4 (AQP4) is the primary cellular water channel in the brain and is abundantly expressed by astrocytes along the blood-brain barrier and brain-cerebrospinal fluid interfaces. Water transport via AQP4 contributes to the activity-dependent volume changes of the extracellular space (ECS), which affect extracellular solute concentrations and neuronal excitability. AQP4 is anchored by α-syntrophin (α-syn), the deletion of which leads to reduced AQP4 levels in perivascular and subpial membranes. We used the real-time iontophoretic method and/or diffusion-weighted magnetic resonance imaging to clarify the impact of α-syn deletion on astrocyte morphology and changes in extracellular diffusion associated with cell swelling in vitro and in vivo. In mice lacking α-syn, we found higher resting values of the apparent diffusion coefficient of water (ADC_W) and the extracellular volume fraction (α). No significant differences in tortuosity (λ) or non-specific uptake (k′), were found between α-syn-negative (α-syn −/−) and α-syn-positive (α-syn +/+) mice. The deletion of α-syn resulted in a significantly smaller relative decrease in α observed during elevated K⁺ (10 mM) and severe hypotonic stress (−100 mOsmol/l), but not during mild hypotonic stress (−50 mOsmol/l). After the induction of terminal ischemia/anoxia, the final values of ADC_W as well as of the ECS volume fraction α indicate milder cell swelling in α-syn −/− in comparison with α-syn +/+ mice. Shortly after terminal ischemia/anoxia induction, the onset of a steep rise in the extracellular potassium concentration and an increase in λ was faster in α-syn −/− mice, but the final values did not differ between α-syn −/− and α-syn +/+ mice. This study reveals that water transport through AQP4 channels enhances and accelerates astrocyte swelling. The substantially altered ECS diffusion parameters will likely affect the movement of neuroactive substances and/or trophic factors, which in turn may modulate the extent of tissue damage and/or drug distribution.     

Aquaporin-4–dependent K+ and water transport modeled in brain extracellular space following neuroexcitation

Potassium (K⁺) ions released into brain extracellular space (ECS) during neuroexcitation are efficiently taken up by astrocytes. Deletion of astrocyte water channel aquaporin-4 (AQP4) in mice alters neuroexcitation by reducing ECS [K⁺] accumulation and slowing K⁺ reuptake. These effects could involve AQP4-dependent: (a) K⁺ permeability, (b) resting ECS volume, (c) ECS contraction during K⁺ reuptake, and (d) diffusion-limited water/K⁺ transport coupling. To investigate the role of these mechanisms, we compared experimental data to predictions of a model of K⁺ and water uptake into astrocytes after neuronal release of K⁺ into the ECS. The model computed the kinetics of ECS [K⁺] and volume, with input parameters including initial ECS volume, astrocyte K⁺ conductance and water permeability, and diffusion in astrocyte cytoplasm. Numerical methods were developed to compute transport and diffusion for a nonstationary astrocyte–ECS interface. The modeling showed that mechanisms b–d, together, can predict experimentally observed impairment in K⁺ reuptake from the ECS in AQP4 deficiency, as well as altered K⁺ accumulation in the ECS after neuroexcitation, provided that astrocyte water permeability is sufficiently reduced in AQP4 deficiency and that solute diffusion in astrocyte cytoplasm is sufficiently low. The modeling thus provides a potential explanation for AQP4-dependent K⁺/water coupling in the ECS without requiring AQP4-dependent astrocyte K⁺ permeability. Our model links the physical and ion/water transport properties of brain cells with the dynamics of neuroexcitation, and supports the conclusion that reduced AQP4-dependent water transport is responsible for defective neuroexcitation in AQP4 deficiency.     

Photosynthesis Regulation by Glucohexaose Through Redox Changes in Cucumis sativus

To investigate the effects of glucohexaose (P6) on cucumber, leaf CO₂ assimilation, chlorophyll fluorescence parameters, chlorophyll content, and carbohydrate metabolism were examined in cucumber plants. The net photosynthetic rate (P _n ) of cucumber leaves was enhanced after being treated with 10 μg mL^?1 P6. The increase was correlated with increases in transpiration rate (E) and stomatal conductance (G _s), whereas the intercellular CO₂ concentration (C _i) was not different from the control plants. Chlorophyll content, absorption of light energy per unit area (ABS/CS), capture of light energy per unit area (TR_o/CS), quantum yield of electron transport per unit area (ET_o/CS), maximum photochemical efficiency of PSII (φP _o), quantum yield of photosynthetic institution electron transfer (φE _o), probability of other electron acceptors that captured exciton-transferred electrons to the electronic chain which exceeds QA (ψ _o), number of reaction centers per unit leaf area (RC/CS_o), and the performance index on absorption basis (PI_ABS) were improved, but heat dissipation per unit area (DI_o/CS) and maximum quantum yield of non-chemical quenching (φD _o) were reduced. In addition, increases in sucrose, soluble sugars, and starch contents were observed in P6-treated plants. However, H₂O₂ scavenger (DMTU) or NADPH oxidase inhibitor (DPI) pretreatment significantly abolished the effect of P6 on photosynthesis. The results demonstrated that ROS played a critical role in P6-induced photosynthesis. The increase in chlorophyll content together with efficient light absorption, transmission, and conversion in P6-treated plants is important for increasing photosynthesis.