Leaf wound induced ultraweak photon emission is suppressed under anoxic stress: Observations of Spathiphyllum under aerobic and anaerobic conditions using novel in vivo methodology

Plants have evolved a variety of means to energetically sense and respond to abiotic and biotic environmental stress. Two typical photochemical signaling responses involve the emission of volatile organic compounds and light. The emission of certain leaf wound volatiles and light are mutually dependent upon oxygen which is subsequently required for the wound-induced lipoxygenase reactions that trigger the formation of fatty acids and hydroperoxides; ultimately leading to photon emission by chlorophyll molecules. A low noise photomultiplier with sensitivity in the visible spectrum (300–720 nm) is used to continuously measure long duration ultraweak photon emission of dark-adapting whole Spathiphyllum leaves (in vivo). Leaves were mechanically wounded after two hours of dark adaptation in aerobic and anaerobic conditions. It was found that (1) nitrogen incubation did not affect the pre-wound basal photocounts; (2) wound induced leaf biophoton emission was significantly suppressed when under anoxic stress; and (3) the aerobic wound induced emission spectra observed was > 650 nm, implicating chlorophyll as the likely emitter. Limitations of the PMT photocathode’s radiant sensitivity, however, prevented accurate analysis from 700–720 nm. Further examination of leaf wounding profile photon counts revealed that the pre-wounding basal state (aerobic and anoxic), the anoxic wounding state, and the post-wounding aerobic state statistics all approximate a Poisson distribution. It is additionally observed that aerobic wounding induces two distinct exponential decay events. These observations contribute to the body of plant wound-induced luminescence research and provide a novel methodology to measure this phenomenon in vivo.

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title: Volatile organic compounds emitted after leaf wounding: On‐line analysis by proton‐transfer‐reaction mass spectrometry
abstract: Volatile organic compounds (VOCs) released from vegetation, including wound-induced VOCs, can have important effects on atmospheric chemistry. The analytical methods for measuring wound-induced VOCs, especially the hexenal family of VOCs (hexenals, hexenols, and hexenyl esters), are complicated by their chemical instability and the transient nature of their formation after leaf and stem wounding. Here we demonstrate that formation and emission of hexenal family compounds can be monitored on-line using proton-transfer-reaction mass spectrometry (PTR-MS), avoiding the need for preconcentration or chromatography. These measurements allow direct analysis of the rapid emission of the parent compound, (Z)-3-hexenal, within 1-2 s of wounding of aspen leaves and then its disappearance and the appearance of its metabolites including (E)-2-hexenal, hexenols, and hexenyl acetates. Similar results were seen in wounded beech leaves and clover. The emission of hexenal family compounds was proportional to the extent of wounding, was not dependent on light, occurred in attached or detached leaves, and was greatly enhanced as detached leaves dried out. Emission of (Z)-3-hexenal from detached drying aspen leaves averaged 500 m gCg 21 (dry leaf weight). Leaf wound compounds were not emitted in a nitrogen atmosphere but were released within seconds of reintroduction of oxygen; this indicates that there are not large pools of hexenyl compounds in leaves. The PTR-MS method also allows the simultaneous detection of less abundant hexanal family VOCs including hexanal, hexanol, and hexyl acetate and VOCs formed in the light (isoprene) or during anoxia (acetaldehyde). PTR- MS may be a useful tool for the analysis of VOC emissions resulting from grazing, herbivory, and other physical damage to vegetation, from harvesting of crops, and from senescing leaves.

title: On the induction of volatile organic compound emissions by plants as consequence of wounding or fluctuations of light and temperature.
abstract: Among the volatile organic compounds (VOCs) emitted by plants, some are characteristic of stress conditions, but their biosynthesis and the metabolic and environmental control over the emission are still unclear. We performed experiments to clarify whether (1) the emission following wounding can occur at distance from the wounding site, from VOC pools subjected to metabolic signals; and (2) the emission of biogenic VOCs generated by membrane damage (e.g. consequent to wounding or ozone exposure) can also be induced by exposure to high light and high temperature, recurrent in nature. In Phragmites australis, leaf cutting caused large and rapid bursts of acetaldehyde both at the cutting site and on parts of the cut leaf distant from the cutting site. This emission was preceded by a transient stomatal opening and did not occur in conditions preventing stomatal opening. This suggests the presence of a large pool of leaf acetaldehyde whose release is under stomatal control. VOCs other than isoprene, particularly acetaldehyde and (E)-2-hexenal, one of the C-6 compounds formed by the denaturation of membrane lipids, were released by leaves exposed to high temperature and high light. The high-temperature treatment (45 degrees C) also caused a rapid stimulation and then a decay of isoprene emission in Phragmites leaves. Isoprene recovered to the original emission level after suspending the high-temperature treatment, suggesting a temporary deficit of photosynthetically formed substrate under high temperature. Emission of C-6 compounds was slowly induced by high temperature, and remained high, indicating that membrane denaturation occurs also after suspending the high-temperature treatment. Conversely, the emission of C-6 compounds was limited to the high-light episode in Phragmites. This suggests that a membrane denaturation may also occur in conditions that do not damage other important plant processes such as the photochemistry of photosynthesis of photoinhibition-insensitive plants. In the photoinhibition-sensitive Arabidopsis thaliana mutant NPQ1, a large but transient emission of (E)-2-hexenal was also observed a few minutes after the high-light treatment, indicating extensive damage to the membranes. However, (E)-2-hexenal emission was not observed in Arabidopsis plants fumigated with isoprene during the high-light treatment. This confirms that isoprene can effectively protect cellular membranes from denaturation. Our study indicates that large, though often transient, VOC emissions by plants occur in nature. In particular, we demonstrate that VOCs can be released by much larger tissues than those wounded and that even fluctuations of light and temperature regularly observed in nature can induce their emissions. This knowledge adds information that is useful for the parameterization of the emissions and for the estimate of biogenic VOC load in the atmosphere.

title: Differential Metabolisms of Green Leaf Volatiles in Injured and Intact Parts of a Wounded Leaf Meet Distinct Ecophysiological Requirements
abstract: Almost all terrestrial plants produce green leaf volatiles (GLVs), consisting of six-carbon (C6) aldehydes, alcohols and their esters, after mechanical wounding. C6 aldehydes deter enemies, but C6 alcohols and esters are rather inert. In this study, we address why the ability to produce various GLVs in wounded plant tissues has been conserved in the plant kingdom. The major product in completely disrupted Arabidopsis leaf tissues was (Z)-3-hexenal, while (Z)-3-hexenol and (Z)-3-hexenyl acetate were the main products formed in the intact parts of partially wounded leaves. 13C-labeled C6 aldehydes placed on the disrupted part of a wounded leaf diffused into neighboring intact tissues and were reduced to C6 alcohols. The reduction of the aldehydes to alcohols was catalyzed by an NADPH-dependent reductase. When NADPH was supplemented to disrupted tissues, C6 aldehydes were reduced to C6 alcohols, indicating that C6 aldehydes accumulated because of insufficient NADPH. When the leaves were exposed to higher doses of C6 aldehydes, however, a substantial fraction of C6 aldehydes persisted in the leaves and damaged them, indicating potential toxicity of C6 aldehydes to the leaf cells. Thus, the production of C6 aldehydes and their differential metabolisms in wounded leaves has dual benefits. In disrupted tissues, C6 aldehydes and their α,β-unsaturated aldehyde derivatives accumulate to deter invaders. In intact cells, the aldehydes are reduced to minimize self-toxicity and allow healthy cells to survive. The metabolism of GLVs is thus efficiently designed to meet ecophysiological requirements of the microenvironments within a wounded leaf.

title: Detection of Plant Volatiles after Leaf Wounding and Darkening by Proton Transfer Reaction “Time-of-Flight” Mass Spectrometry (PTR-TOF)
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title: Volatile organic compounds emanating from indoor ornamental plants.
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title: Green leaf volatiles: biosynthesis, biological functions and their applications in biotechnology.
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title: Plants Attract Parasitic Wasps to Defend Themselves against Insect Pests by Releasing Hexenol
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title: A look at some systemic properties of self-bioluminescent emission
abstract: Self-bioluminescent emission (SBE) is a type of biological chemiluminescence where photons are emitted as part of chemical reactions occurring during metabolic processes. This emission is also known as biophoton emission, ultraweak photon emission and ultraweak bioluminescence. This paper outlines research over the past century on some systemic properties of SBE as measured with biological detectors, photomultiplier detectors and ultra-sensitive imaging arrays. There is an apparent consensus in the literature that emission in the deep blue and ultraviolet (150-450nm) is related to DNA / RNA processes while emission in the red and near infrared (600-1000nm) is related to mitochondria and oxidative metabolisms involving reactive oxygen species, singlet oxygen and free radicals in plant, animal and human cells along with chlorophyll fluorescent decay in plants. Additionally, there are trends showing that healthy, unstressed and uninjured samples have less emission than samples that are unhealthy, stressed or injured. Mechanisms producing this emission can be narrowed down by isolating the wavelength region of interest and waiting for short-term fluorescence to decay leaving the ultraweak long-term metabolic emission. Examples of imaging this emission in healthy versus unhealthy, stressed versus unstressed, and injured versus uninjured plant parts are shown. Further discussion poses questions still to be answered related to properties such as coherence, photon statistics, and methodological means of isolating mechanisms.

title: Properties of biophotons and their theoretical implications.
abstract: The word "biophotons" is used to denote a permanent spontaneous photon emission from all living systems. It displays a few up to some hundred photons/(s x cm2) within the spectral range from at least 260 to 800 nm. It is closely linked to delayed luminescence (DL) of biological tissues which describes the long term and ultra weak reemission of photons after exposure to light illumination. During relaxation DL turns continuously into the steady state biophoton emission, where both, DL and biophoton emission exhibit mode coupling over the entire spectrum and a Poissonian photo count distribution. DL is representing excited states of the biophoton field. The physical properties indicate that biophotons originate from fully coherent and sometimes even squeezed states. The physical analysis provides thermodynamic and quantum optical interpretation, in order to understand the biological impacts of biophotons. Biological phenomena like intracellular and intercellular communication, cell growth and differentiation, interactions among biological systems (like "Gestaltbildung" or swarming), and microbial infections can be understood in terms of biophotons. "Biophotonics", the corresponding field of applications, provide a new powerful tool for assessing the quality of food (like freshness and shelf life), microbial infections, environmental influences and for substantiating medical diagnosis and therapy.

title: Plant chemiluminescence.
abstract: Light production by plants was confirmed by measuring chemiluminescence from root and stem tissue of peas (Pisum sativum), beans (Phaseolus vulgaris), and corn (Zea mays) in a modified scintillation spectrophotometer. Chemiluminescence was inhibited by treating pea roots with boiling ethanol or by placing them in a N(2) gas phase. Chemiluminescence was increased by an O(2) gas phase or by the addition of luminol. NaN(3) and NaCN blocked both in vitro and in vivo chemiluminescence.It is postulated that the source of light is the hydrogen peroxide-peroxidase enzyme system. It is known that this system is responsible for chemiluminescence in leukocytes and it seems likely that a similar system occurs in plants.

title: Role of reactive oxygen species in ultra-weak photon emission in biological systems.
abstract: Ultra-weak photon emission originates from the relaxation of electronically excited species formed in the biological systems such as microorganisms, plants and animals including humans. Electronically excited species are formed during the oxidative metabolic processes and the oxidative stress reactions that are associated with the production of reactive oxygen species (ROS). The review attempts to overview experimental evidence on the involvement of superoxide anion radical, hydrogen peroxide, hydroxyl radical and singlet oxygen in both the spontaneous and the stress-induced ultra-weak photon emission. The oxidation of biomolecules comprising either the hydrogen abstraction by superoxide anion and hydroxyl radicals or the cycloaddition of singlet oxygen initiate a cascade of oxidative reactions that lead to the formation of electronically excited species such as triplet excited carbonyl, excited pigments and singlet oxygen. The photon emission of these electronically excited species is in the following regions of the spectrum (1) triplet excited carbonyl in the near UVA and blue-green areas (350-550nm), (2) singlet and triplet excited pigments in the green-red (550-750nm) and red-near IR (750-1000nm) areas, respectively and (3) singlet oxygen in the red (634 and 703nm) and near IR (1270nm) areas. The understanding of the role of ROS in photon emission allows us to use the spontaneous and stress-induced ultra-weak photon emission as a non-invasive tool for monitoring of the oxidative metabolic processes and the oxidative stress reactions in biological systems in vivo, respectively.

title: Ultra-weak photon emission from biological samples: definition, mechanisms, properties, detection and applications.
abstract: This review attempts to summarize molecular mechanisms, spectral and intensity properties, detection techniques and applications of ultra-weak photon emission. Ultra-weak photon emission is the chemiluminescence from biological systems where electronically excited species are formed during oxidative metabolic or oxidative stress processes. It is generally accepted that photons are emitted (1) at near UVA, visible, and near IR spectral ranges from 350 to 1300nm and (2) at the intensity of photon emission in the range of several units to several hundreds (oxidative metabolic process) and several hundreds to several thousands (oxidative stress process) photons s(-1)cm(-2). Current development in detection using low-noise photomultiplier tubes and imaging using highly sensitive charge coupled device cameras allows temporal and spatial visualization of oxidative metabolic or oxidative stress processes, respectively. As the phenomenon of ultra-weak photon emission reflects oxidative metabolic or oxidative stress processes, it can be widely used as a non-invasive tool for monitoring of the physiological state of biological systems.

title: Using spontaneous photon emission to image lipid oxidation patterns in plant tissues.
abstract: Plants, like almost all living organisms, spontaneously emit photons of visible light. We used a highly sensitive, low-noise cooled charge coupled device camera to image spontaneous photon emission (autoluminescence) of plants. Oxidative stress and wounding induced a long-lasting enhancement of plant autoluminescence, the origin of which is investigated here. This long-lived phenomenon can be distinguished from the short-lived chlorophyll luminescence resulting from charge recombinations within the photosystems by pre-adapting the plant to darkness for about 2 h. Lipids in solvent were found to emit a persistent luminescence after oxidation in vitro, which exhibited the same time and temperature dependence as plant autoluminescence. Other biological molecules, such as DNA or proteins, either did not produce measurable light upon oxidation or they did produce a chemiluminescence that decayed rapidly, which excludes their significant contribution to the in vivo light emission signal. Selective manipulation of the lipid oxidation levels in Arabidopsis mutants affected in lipid hydroperoxide metabolism revealed a causal link between leaf autoluminescence and lipid oxidation. Addition of chlorophyll to oxidized lipids enhanced light emission. Both oxidized lipids and plants predominantly emit light at wavelengths higher than 600 nm; the emission spectrum of plant autoluminescence was shifted towards even higher wavelengths, a phenomenon ascribable to chlorophyll molecules acting as luminescence enhancers in vivo. Taken together, the presented results show that spontaneous photon emission imaged in plants mainly emanates from oxidized lipids. Imaging of this signal thus provides a simple and sensitive non-invasive method to selectively visualize and map patterns of lipid oxidation in plants.

title: Linoleic Acid-Induced Ultra-Weak Photon Emission from Chlamydomonas reinhardtii as a Tool for Monitoring of Lipid Peroxidation in the Cell Membranes
abstract: Reactive oxygen species formed as a response to various abiotic and biotic stresses cause an oxidative damage of cellular component such are lipids, proteins and nucleic acids. Lipid peroxidation is considered as one of the major processes responsible for the oxidative damage of the polyunsaturated fatty acid in the cell membranes. Various methods such as a loss of polyunsaturated fatty acids, amount of the primary and the secondary products are used to monitor the level of lipid peroxidation. To investigate the use of ultra-weak photon emission as a non-invasive tool for monitoring of lipid peroxidation, the involvement of lipid peroxidation in ultra-weak photon emission was studied in the unicellular green alga Chlamydomonas reinhardtii. Lipid peroxidation initiated by addition of exogenous linoleic acid to the cells was monitored by ultra-weak photon emission measured with the employment of highly sensitive charged couple device camera and photomultiplier tube. It was found that the addition of linoleic acid to the cells significantly increased the ultra-weak photon emission that correlates with the accumulation of lipid peroxidation product as measured using thiobarbituric acid assay. Scavenging of hydroxyl radical by mannitol, inhibition of intrinsic lipoxygenase by catechol and removal of molecular oxygen considerably suppressed ultra-weak photon emission measured after the addition of linoleic acid. The photon emission dominated at the red region of the spectrum with emission maximum at 680 nm. These observations reveal that the oxidation of linoleic acid by hydroxyl radical and intrinsic lipoxygenase results in the ultra-weak photon emission. Electronically excited species such as excited triplet carbonyls are the likely candidates for the primary excited species formed during the lipid peroxidation, whereas chlorophylls are the final emitters of photons. We propose here that the ultra-weak photon emission can be used as a non-invasive tool for the detection of lipid peroxidation in the cell membranes.

title: Lipoxygenase in singlet oxygen generation as a response to wounding: in vivo imaging in Arabidopsis thaliana
abstract: Wounding, one of the most intensive stresses influencing plants ontogeny and lifespan, can be induced by herbivory as well as by physical factors. Reactive oxygen species play indispensable role both in the local and systemic defense reactions which enable “reprogramming” of metabolic pathways to set new boundaries and physiological equilibrium suitable for survival. In our current study, we provide experimental evidence on the formation of singlet oxygen (1O2) after wounding of Arabidopsis leaves. It is shown that 1O2 is formed by triplet-triplet energy transfer from triplet carbonyls to molecular oxygen. Using lipoxygenase inhibitor catechol, it is demonstrated that lipid peroxidation is initiated by lipoxygenase. Suppression of 1O2 formation in lox2 mutant which lacks chloroplast lipoxygenase indicates that lipoxygenase localized in chloroplast is predominantly responsible for 1O2 formation. Interestingly, 1O2 formation is solely restricted to chloroplasts localized at the wounding site. Data presented in this study might provide novel insight into wound-induced signaling in the local defense reaction.

title: Arabidopsis lipoxygenase 2 is essential for formation of green leaf volatiles and five‐carbon volatiles
abstract: Plants biosynthesize a variety of bioactive lipid derivatives, such as green leaf volatiles (GLVs) and jasmonates (JAs). Here we identify a lipoxygenase 2 (LOX2) involved in GLV biosynthesis in Arabidopsis using mutant lines for each of the six LOX isoforms present in Arabidopsis. We found that formation of five carbon volatiles was also dependent on LOX2. LOX2 is known to be involved in formation of JA; thus, LOX2 is apparently versatile in function. The results in this study suggested that LOX2 activity is suppressed in intact cells but activated upon tissue damage to support the rapid GLV‐burst observed in wounded leaves.

title: Imaging the production of singlet oxygen in vivo using a new fluorescent sensor, Singlet Oxygen Sensor Green.
abstract: Singlet oxygen is known to be produced by cells in response to photo-oxidative stresses and wounding. Due to singlet oxygen being highly reactive, it is thought to have a very short half-life in biological systems and, consequently, it is difficult to detect. A new commercially available reagent (singlet oxygen sensor green, SOSG), which is highly selective for singlet oxygen, was applied to a range of biological systems that are known to generate singlet oxygen. Induction of singlet oxygen production by the addition of myoglobin to liposome preparations demonstrated that the singlet oxygen-induced increases in SOSG fluorescence closely followed the increase in the concentration of conjugated dienes, which is stoichiometrically related to singlet oxygen production. Applications of photo-oxidative stresses to diatom species and leaves, which are known to result in the production of singlet oxygen, produced large increases in SOSG fluorescence, as did the addition of 3-(3',4'-dichlorophenyl)1,1-dimethylurea (DCMU) to these systems, which inhibits electron transport in photosystem II and stimulates singlet oxygen production. The conditional fluorescent (flu) mutant of Arabidopsis produces singlet oxygen when exposed to light after a dark period, and this coincided with a large increase in SOSG fluorescence. Wounding of leaves was followed by an increase in SOSG fluorescence, even in the dark. It is concluded that SOSG is a useful in vivo probe for the detection of singlet oxygen.

title: Light as both an input and an output of wound-induced reactive oxygen formation in Arabidopsis leaves
abstract: The wound response of plants is characterised by rapid changes in gene expression, biochemistry and physiology, and is important both in its own right and as a model for studying events elicited by herbivory. We have recently identified links between light and the wound response in Arabidopsis leaves. This includes an influence of the external light environment on the molecular and biochemical response to wounding, and the observation that endogenous bioluminescence (light emission) is a consequence of tissue damage. Here, we show that this link extends to the production of reactive oxygen species. We show that wounding causes rapid, light-dependent production of superoxide and hydrogen peroxide in chloroplasts via disruption of photosynthesis, and that wound-induced bioluminescence is a consequence of the generation of singlet oxygen.

title: Spontaneous ultraweak bioluminescence in plants: origin, mechanisms and properties.
abstract: An analysis of theoretical knowledge and experimental results of ultraweak luminescence (UWL) is provided. The role of excited state of molecules and free radicals, formed in various biochemical reactions, in UWL is discussed. UWL of model reactions and in vivo systems are compared. The hypothesis of coherent electromagnetic field as a source of UWL is also discussed. Spectral, kinetic and temporal properties of UWL are summarised, as well as their connection with its origin and role in the organism. Attention is paid to recent progress in experimental methods of low-light detection. The possible use of UWL in environmental studies, selection and other applications is discussed.

title: INDUCTION OF A TRANSIENT ENHANCEMENT OF LOW LEVEL CHEMILUMINESCENCE IN INTACT LEAVES BY ANAEROBIC TREATMENT
abstract: Intrinsic low level chemiluminescence of dark adapted intact leaves exhibits a transient enhancement of light emission when ambient air is replaced with an anaerobic atmosphere. Re establishing aerobic conditions gives rise to a complex kinetic behavior of the light emission in the post‐anoxic phase. Metabolic changes within the leaf are suggested to be responsible for this phenom enon, where intactness of the leaf is found to be of crucial importance. Leaf homogenate showed nearly oxygen independent chemiluminescence. In contrast, low level chemiluminescence of UV‐light damaged leaves and of chloroplast preparations was inhibited by oxygen depletion. Spectral analyses of the ultraweak light emission from the intact leaf indicates that in both aerobic and anaerobic cases, the source of the ultraweak light is the same. The similarity to the spectrum of delayed fluorescence also obtained from the leaf suggests that chlorophyll is the light emitting species.

title: Plant Anaerobic Stress as a Novel Trend in Ecological Physiology, Biochemistry, and Molecular Biology: 1. Establishment of a New Scientific Discipline
abstract: This review attempted to follow the establishment of a novel branch of biology arisen at the interfaces between plant physiology, biochemistry, and molecular biology—plant anaerobic stress. Most attention was given to the early period of these investigations, the activity of the members of International Society for Plant Anaerobiosis in particular, and the contribution of Russian scientists, who played a significant role at that time in the establishment and international recognition of this new trend. In this connection, the following points are considered: (1) Crawford's metabolic theory, which could not withstand experimental verification but induced an active discussion, thus stimulating further investigations in this field; (2) a concept of two main strategies of plant adaptation to anaerobic stress (true and apparent adaptation), which was put forward based on the following experimental data: (a) a discovery of a paradoxical phenomenon of hyper-sensitivity, but not hyper-resistance to anoxia, of the flood-tolerant plant roots (“apparent” tolerance); (b) the elucidation of the physiological role of oxygen transported from aerated organs of flood-tolerant plants to the roots inhabiting anaerobic environment; (c) demonstration of the key role of both energy metabolism, and (d) substrate providing for glycolysis and ethanolic fermentation in plants manifesting “true” tolerance to oxygen deprivation; (3) the discovery of plant stress proteins; and finally (4) pH-stat theory put forward by Davies.

title: BIOPHOTON EMISSION: EXPERIMENTAL BACKGROUND AND THEORETICAL APPROACHES
abstract: Biophoton emission is a general phenomenon of living systems. It concerns a weak photon radiation from a few to some hundred photons per second, per square centimeter surface area, at least within the spectral region from 200 to 800 nm. The results indicate that biophoton emission can be assigned to a coherent field within living organisms, its functions being intra and intercellular regulation and communication. This review deals with some central results and their interpretation.

title: Detection of Photon Emission from Biological Systems
abstract: Since photon emission from living organisms is extremely weak (the photon flux is the order of less than 10−15 W) in comparison with a normal light source, some very sensitive methods and devices have been established to detect biophotons both in integral emission intensity and the two-dimensional pattern of the emission on living organisms. This chapter will discuss the modern technology and systems used by various research groups to perform the detection, spectral analysis and two-dimensional imaging of ultraweak photon emission from living organisms such as cells, tissues and even organs. In addition, photon emission from biological system has some intrinsic features, such as coherence, differing with that from non-living systems. Thus, some particular methods are needed to characterize the physical and biological features of photon emission from biological systems.

title: Ultraweak and Induced Photon Emission After Wounding of Plants
abstract: The ultraweak and induced photon emission were measured by a single photon counting equipment (Photomultiplier Hamamatsu R562) on Cucurbita pepo variaca styriacae after wounding. Wounding significantly changes the emission from a stationary to a nonstationary state and the shape of the decay curve obtained after light illumination. The rise in the ultraweak photon emission depends on the kind of wounding and its localization on the plant. The decay curves obtained after wounding could be better fit by an exponential function than by a hyperbolic one. So the biophoton emission correlates with physiological and bioelectrical changes like membrane depolarizations as they also depend on the kind of injury.

title: Research on the Ultraweak Photon Emission from Anti-cancer Plants
abstract: UPE (Ultraweak photon emission) is one kind of a common phenomenon in biological organisms. It contains a wealth of information of biological functions. In this paper, single photon counting system is used to measure UPE of some anticancer herbal plants. For the plants, the changes of UPE under different water condition are studied and the varying laws of ultraweak photon number with time are analysed. The results are higher fit double exponential decay law.

title: Chlorophyll fluorescence emission spectrum inside a leaf.
abstract: Chlorophyll a fluorescence can be used as an early stress indicator. Fluorescence is also connected to photosynthesis so it can be proposed for global monitoring of vegetation status from a satellite platform. Nevertheless, the correct interpretation of fluorescence requires accurate physical models. The spectral shape of the leaf fluorescence free of any re-absorption effect plays a key role in the models and is difficult to measure. We present a vegetation fluorescence emission spectrum free of re-absorption based on a combination of measurements and modelling. The suggested spectrum takes into account the photosystem I and II spectra and their relative contribution to fluorescence. This emission spectrum is applicable to describe vegetation fluorescence in biospectroscopy and remote sensing.

title: Chlorophyll Fluorescence in Plant Biology
abstract: Several molecules absorb light energy which they emit after a time difference (lifetime) as radiation energy. Molecules remain at a low energy level or the ground electronic singlet state (So) or the lowest vibrational level at room temperature (Noomnarm and Clegg, 2009). On absorption of a photon, the molecule is excited from So to the first electronic excited singlet state S1 within < 10-15 s-1 . These molecules can also be transferred to higher energy levels (S2 to Sn) also. These excited state molecules can relax to the S1 electronic state via vibrational relaxation within 10-12 s-1. The molecule will ultimately relax to the So state through photon emission, which is called fluorescence emission. Also here, the energy of the emitted photon must equal the changes in the energy levels.

title: Re-absorption of chlorophyll fluorescence in leaves revisited. A comparison of correction models.
abstract: The application of correction methods to account for re-absorption of chlorophyll fluorescence emission in leaves is subject to a number of controversies in the literature. These uncertainties lead to high discrepancies in the corrected spectral distribution of fluorescence and consequently in the interpretation of related physiological features of plants, according to the chosen method used in the process of correction. In this research, three correction methods, based on transmittance and/or reflectance measurements on leaves, were analysed comparatively. One method gave high values for the corrected fluorescence ratio between 685 nm and 737 nm (F685/F737 approximately 7 to 20 according to the different species of leaves). The two other methods were found to give similar results with corrected fluorescence ratios around a value of two (F685/F737 approximately 2). While the first method was developed in the light of empirical considerations, the latter two models are based upon defined physical approaches depicting interaction between light and matter. The theoretical basis of these methods, the validation methodologies used to support them and the similarity in the spectra corrected by light re-absorption for both models, all showed that they should be treated as confident and suitable approximations to solve the problem of light re-absorption in leaves.

title: Reviewing the relevance of fluorescence in biological systems.
abstract: Fluorescence is emitted by diverse living organisms. The analysis and interpretation of these signals may give information about their physiological state, ways of communication among species and the presence of specific chemicals. In this manuscript we review the state of the art in the research on the fluorescence emitted by plant leaves, fruits, flowers, avians, butterflies, beetles, dragonflies, millipedes, cockroaches, bees, spiders, scorpions and sea organisms and discuss its relevance in nature.

title: Experimental study on photocount statistics of the ultraweak photon emission from some living organisms
abstract: The hypothesis that biophotons display a high degree of coherence was tested by measuring photocount statistics (PCS) of the ultraweak photon emission from three living organisms (cucumber seedling, mungbean seedling and soybean rhizobium bacteroids) with a high-sensitivity single-photon counter. For comparison, the same experiments were performed for laser beam, randomized laser beam, chemiluminescence from autoxidation of luminol and the dark counts of the equipment. Photocount distributions, close to Poissonian, were observed for the three tested biological systems but not for the pure chemiluminescence of luminol.

title: Physical aspects of biophotons
abstract: By comparing the theoretically expected results of photon emission from a chaotic (thermal) field and those of an ordered (fully coherent) field with the actual experimental data, one finds ample indications for the hypothesis that ‘biophotons’ originate from a coherent field occurring within living tissues. A direct proof may be seen in the hyperbolic relaxation dynamics of spectral delayed luminescence under ergodic conditions. A possible mechanism has to be founded on Einstein's balance equation and, under stationary conditions, on energy conservation including a photochemical potential. It is shown that the considered equations deliver, besides the thermal equilibrium, a conditionally stable region far away from equilibrium, which can help to describe both ‘biophoton emission’ and biological regulation.

title: Coherent nature of the radiation emitted in delayed luminescence of leaves
abstract: After exposure to light, a living system emits a photon signal of characteristic shape. The signal has a small decay region and a long tail region. The flux of photons in the decay region changes by 2 to 3 orders of magnitude, but remains almost constant in the tail region. The decaying part is attributed to delayed luminescence and the constant part to ultra-weak luminescence. Biophoton emission is the common name given to both kinds of luminescence, and photons emitted are called biophotons. The decay character of the biophoton signal is not exponential, which is suggestive of a coherent signal. We sought to establish the coherent nature by measuring the conditional probability of zero photon detection in a small interval Delta. Our measurements establish the coherent nature of biophotons emitted by different leaves at various temperatures in the range 15-50 degrees C. Our set up could measure the conditional probability for Delta</=100 &mgr;s in only 100 ms, which enabled us to make its measurement in the decaying part of the signal. Various measurements were repeated 2000 times in contiguous intervals, which determined the dependence of the conditional probability on signal strength. The observed conditional probabilities at different signal strengths are in agreement with the predictions for coherent photons. The agreement is impressive at the discriminatory range, 0.1-5 counts per Delta, of signal strengths. The predictions for coherent and thermal photons differ substantially in this range. We used the values of Delta in the range, 10 &mgr;s-10 ms for obtaining a discriminatory signal strength in different regions of a decaying signal. These measurements establish the coherent nature of photons in all regions of a biophoton signal from 10 ms to 5 hr. We have checked the efficacy of out method by measuring the conditional probability of zero-photon detection in the radiation of a light emitting diode along with a leaf for Delta in the range 10 &mgr;s-100 &mgr;s. The conditional probability in the diode radiation was different from the prediction for coherent photons when the signal strength was less than 2.5 counts per Delta. Only the diode radiation exhibited photon bunching at signal strength around 0.05 count in Delta.

title: Energy transfer dynamics of an isolated light harvesting complex of Photosystem I from spinach: time-resolved fluorescence measurements at 295 K and 77 K
abstract: Time-resolved fluorescence relaxation measurements have been performed at 295 K and 77 K on a preparation of the light-harvesting complex of Photosystem I (LHCI) containing about 100 chlorophylls (Chl) per complex. At both temperatures five exponential components were required to fit the data. The resulting determined lifetimes are 30 ps, 200 ps, 1.0 ns, 3.3 ns and 6.5 ns. Previous steady-state fluorescence measurements demonstrated that the emission at 735 nm increases dramatically upon cooling to 77 K. Time-resolved fluorescence measurements performed at 77 K reveal the presence of two risetimes for emission between 700 and 750 nm. These results are consistent with an efficient trap for electronic excitation existing at longer wavelengths than the absorption maximum of P700, the reaction center of Photosystem I (PS I). Excitation of Chl b results in a relative amplitude enhancement of decay components emitting between 720 and 750 nm. This confirms that Chl b is specifically associated with long wavelength emitters in Photosystem I.

title: High resolution emission spectra of one second delayed fluorescence from chloroplasts
abstract: The first well resolved emission spectra of white light‐illuminated spinach chloroplasts at room temperature show that one second delayed fluorescence occurs at 685 nm. We demonstrate that reabsorption of this delayed fluorescence induces the second (probably prompt) emission observed at 730 nm and which we identify with the photosystem I peripheral antenna system.

title: The distribution and possible origin of blue-green fluorescence in control and stressed barley leaves.
abstract: Apart from red (chlorophyll a) fluorescence, ultraviolet (UV) excitation of plant leaves results in blue-green intrinsic light emission. In order to analyse the potentially modifying influence of stress on this blue-green fluorescence (BGF), and to determine its origin, the fluorescence was characterised by spectroscopy, and two and three dimensional microscopy in leaves of Hordeum vulgare L. under UV and drought stress. Leaves of wild-type and proanthocyanidin-free mutant barley were compared. In general, the BGF was enhanced by stress. The analysis of the distribution of BGF pointed to differences in response to the different stress conditions and served to indicate possible sources of the fluorescence. This study showed that (1) the selective enhancement of BGF by supplementary UV radiation (280-360 nm) in upper leaf tissues of wild-type but not in proanthocyanidin-free leaves, pointed to flavonoids as possible emitters; (2) drought-inducible BGF emission was found in cell walls and to a smaller extent in upper leaf tissue regardless of genotype, thus independent of impairment in flavonoid biosynthesis and (3) intracellular BGF, which co-localised with red chlorophyll fluorescence, was inducible in mesophyll cells of both genotypes by short-term application of high levels of UV radiation.

title: Chlorophyll fluorescence imaging of photosynthetic activity with the flash-lamp fluorescence imaging system
abstract: A flash-lamp chlorophyll (Chl) fluorescence imaging system (FL-FIS) is described that allows to screen and image the photosynthetic activity of several thousand leaf points (pixels) of intact leaves in a non-destructive way within a few seconds. This includes also the registration of several thousand leaf point images of the four natural fluorescence bands of plants in the blue (440 nm) and green (520 nm) regions as well as the red (near 690 nm) and far-red (near 740 nm) Chl fluorescence. The latest components of this Karlsruhe FL-FIS are presented as well as its advantage as compared to the classical single leaf point measurements where only the fluorescence information of one leaf point is sensed per each measurement. Moreover, using the conventional He-Ne-laser induced two-wavelengths Chl fluorometer LITWaF, we demonstrated that the photosynthetic activity of leaves can be determined measuring the Chl fluorescence decrease ratio, RFd (defined as Chl fluorescence decrease Fd from maximum to steady state fluorescence Fs:Fd/Fs), that is determined by the Chl fluorescence induction kinetics (Kautsky effect). The height of the values of the Chl fluorescence decrease ratio RFd is linearly correlated to the net photosynthetic CO2 fixation rate PN as is indicated here for sun and shade leaves of various trees that considerably differ in their PN. Imaging the RFd-ratio of intact leaves permitted the detection of considerable gradients in photosynthetic capacity across the leaf area as well as the spatial heterogeneity and patchiness of photosynthetic quantum conversion within the control leaf and the stressed plants. The higher photosynthetic capacity of sun versus shade leaves was screened by Chl fluorescence imaging. Profile analysis of fluoresence signals (along a line across the leaf area) and histograms (the signal frequency distribution of the fluorescence information of all measured leaf pixels) of Chl fluorescence yield and Chl fluorescence ratios allow, with a high statistical significance, the quantification of the differences in photosynthetic activity between various areas of the leaf as well as between control leaves and water stressed leaves. The progressive uptake and transfer of the herbicide diuron via the petiole into the leaf of an intact plant and the concomitant loss of photosynthetic quantum conversion was followed with high precision by imaging the increase of the red Chl fluorescence F690. Differences in the availability and absorption of soil nitrogen of crop plants can be documented via this flash-lamp fluorescence imaging technique by imaging the blue/red ratio image F440/F690, whereas differences in Chl content are detected by collecting images of the fluorescence ratio red/far-red, F690/F740.

title: Ultra-weak photon emission from biological samples: definition, mechanisms, properties, detection and applications.
abstract: This review attempts to summarize molecular mechanisms, spectral and intensity properties, detection techniques and applications of ultra-weak photon emission. Ultra-weak photon emission is the chemiluminescence from biological systems where electronically excited species are formed during oxidative metabolic or oxidative stress processes. It is generally accepted that photons are emitted (1) at near UVA, visible, and near IR spectral ranges from 350 to 1300nm and (2) at the intensity of photon emission in the range of several units to several hundreds (oxidative metabolic process) and several hundreds to several thousands (oxidative stress process) photons s(-1)cm(-2). Current development in detection using low-noise photomultiplier tubes and imaging using highly sensitive charge coupled device cameras allows temporal and spatial visualization of oxidative metabolic or oxidative stress processes, respectively. As the phenomenon of ultra-weak photon emission reflects oxidative metabolic or oxidative stress processes, it can be widely used as a non-invasive tool for monitoring of the physiological state of biological systems.

title: Biophoton interaction in biological systems: evidence of photonic info-energy transfer?
abstract: Photons are continuously absorbed and emitted by all living cells. A possible means of releasing energy when an electron changes energy states during a biochemical reaction is via biophoton emission. An example of energy transfer in biological systems is the process of photosynthesis. Biophoton emission has also been proposed as one possible mechanism responsible for intra- and intercellular communication (information transfer) as well as for regulation of biological and biochemical functions within cells and living systems. Measurements by other researchers of this emission have shown it has the properties of coherent light and is measurable from the UV through the near IR. Experimental evidence gathered by various researchers since the 1920's indicates that light plays an important role in certain biological functions and processes. Through a series of experiments we have observed resonance effects between plant parts measured using a highly sensitive, low noise, cooled CCD in total darkness in a light-tight chamber. Dynamical systems theory offers a plausible explanation for resonance effects we have observed. The role of photonic interaction at the systemic level in biological systems has received relatively little attention. Yet, a better understanding of these processes would help us in deciphering the nature and role of light in biological systems.

title: Photoacclimation in spathiphyllum.
abstract: We studied photoacclimation in Spathiphyllum grown at an irradiance of 40 or 420 micromol/m2 s (LL or HL, respectively). All parameters studied responded to acclimation. Leaves at LL, in contrast to HL, were thinner and oriented perpendicular to the incident light, had more chlorophyll per g f w, fewer stomata on the upper leaf surface and a reduced layer of mesophyll cells. Their chloroplasts at HL had wider grana with less thylakoids per granum, and better organized photosystems than at LL. PSI and PSII activities per mg chlorophyll ( Vmax ), and PSI and PSII content (total activity per g f w), were lower at LL than at HL and so was the light requirement for saturation of the PSI or PSII partial photoreactions, suggesting that fewer photosystems with larger antenna size prevail at LL, but many more with smaller antenna size at HL. Analysis of chlorophyll distribution among the thylakoid pigment-protein complexes showed less antenna chlorophyll serving PSII (CPa+LHCP1+LHCP3) than that serving PSI (CPIa+CPI+LHCP2) at LL as compared to HL, and thus a lower PSII/PSI ratio at LL, in agreement with the general finding that LL plants, with larger PSII antenna size, have lower PSII/PSI ratio. The increase in PSI antenna size at LL was correlated with the increase in the distribution of chlorophyll in pigment-protein complexes serving PSI, and a very large chlorophyll/protein molar ratio in the isolated CPI complex. On the other hand, the PSII antenna chlorophyll (CPa+LHCP1+LHCP3) on a g f w basis, and the chlorophyll a/b ratio remained more or less constant at LL or HL. This may reflect our finding that Spathiphyllum contains mainly the 27 kDa inner LHCII antenna protein, the size of which remains unaffected by photoacclimation. The increase in the distribution of chlorophyll in pigment-protein complexes serving PSII at HL, therefore, reflects the higher population of PSII at HL. Very high PSI activity was found at HL, which we attribute to the highly organized small in size PSI.