Photodynamic Stress Research Articles

Glycine max (L.) Merr. (Soybean) metabolome responses to potassium availability

Potassium (K+) has vital physiological and metabolic functions in plants and its availability can impact tolerance to biotic and abiotic stress conditions. Limited studies have investigated the effect of K+ fertilization on soybean metabolism. Using integrated omics, ionomics and metabolomics, we investigated the field-grown Glycine max (soybean) response, after four K+ soil fertilization rates. Soybean leaf and pod tissue (valves and immature seeds) extracts were analysed by ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS) and inductively coupled plasma optical emission spectroscopy (ICP-OES). Multivariate analyses (PCA-X&Y e O2PLS-DA) showed that 51 compounds of 19 metabolic pathways were regulated in response to K+ availability. Under very low potassium availability, soybean plants accumulated of Ca2+, Mg2+, Fe2+, Cu2+, and B in young and old leaves. Potassium fertilization upregulated carbohydrate, galactolipid, and flavonol glycoside biosynthesis in leaves and pod valves, while K+ deficient pod tissues showed increasing amino acids, oligosaccharides, benzoic acid derivatives, and isoflavones contents. Severely K+ deficient soils elicited isoflavones, coumestans, pterocarpans, and soyasaponins in trifoliate leaves, likely associated to oxidative and photodynamic stress status. Additionally, results demonstrate that L-asparagine content is higher in potassium deficient tissues, suggesting this compound as a biomarker of K+ deficiency in soybean plants. These results demonstrate that potassium soil fertilization did not linearly contribute to changes in specialised constitutive metabolites of soybean. Altogether, this work provides a reference for improving the understanding of soybean metabolism as dependent on K+ availability.

ζ-Carotene: Generation and Quenching of Singlet Oxygen, Comparison with Phytofluene.

It is known that C40 carotenoids with a short chain of conjugated double bonds (CDB) (5 and 7, respectively) are universal precursors in the biosynthesis of colored carotenoids in plant cells. Previously, using mainly stationary measurements of photosensitized phosphorescence of singlet oxygen (1O2), we discovered that phytofluene efficiently generates 1O2 in aerated solution and therefore, can serve as a source of the UV photodynamic stress in living cells [Ashikhmin et al., Biochemistry (Moscow), 2020, 85, 773]. In the present paper, by using novel pulsed light emitting diodes (LEDs), aerated hexafluorobenzene as a solvent and time-resolved measurements of 1O2 phosphorescence we confirmed that phytofluene efficiently photosensitizes 1O2 formation. The quantum yield of this process according to the novel experiments is about 0.4. An ability to generate 1O2 was also found in aerated solutions of ζ-carotene although the quantum yield of this process is 30-fold lower that in phytofluene solutions. Both carotenoids were found to quench 1O2 in the dark with the quenching rate constants equal to (3.6 ± 0.9)×107 and (2.1 ± 0.2)×108 M-1s-1, respectively. To our knowledge, the rate constant of 1O2 quenching by ζ-carotene has been reported in the present paper for the first time. It follows from the data obtained that the rate constants of 1O2 quenching by both carotenoids are much (by 2-3 orders of magnitude) smaller than the rate constant of the diffusion-limited biomolecular reactions. Hence, both carotenoids are weak protectors against 1O2 oxidative activity. It is more likely that they are potential promoters of photodynamic stress in living cells.

The oxidative stress, mitochondrial pathway apoptosis, and the antagonistic effects of chrysophanol in SH-SY5Y cells via DTPP-induced photodynamic therapy

BackgroundTo investigate the susceptibility of SH-SY5Y cells to DTPP-based photodynamic therapy (PDT) and the antagonistic effects of chrysophanol (Chr) on PDT. MethodsPDT photocytotoxicity to cells was quantified and determined by exposing increasing concentrations of DTPP between 2.5 to 20 μg/mL to radiation with energy densities of 1.2–9.6 J/cm2 at 630-nm wavelength. Sodium azide (SA, NaN3) and d-mannitol (DM) were employed to study the reaction type of PDT. The photodynamic stress after PDT was assessed by superoxide dismutase (SOD), malondialdehyde (MDA), and total antioxidative capacity (T-AOC) assays. The apoptosis pathway of SH-SY5Y cells after PDT was studied by the determination of JC-1 and caspase-9/Caspase-3 concentrations. MTT and double fluorescence staining assays were applied to study the effect of Chr on cell survival and apoptosis rate in PDT, respectively. PI was used to detect the effect of Chr on cell membrane integrity after DTPP-PDT treatment. ResultsThe dose-dependent killing effect of high DTPP concentrations and irradiation doses were identified. Cell apoptosis is mediated by a mitochondrial pathway with a total apoptosis rate of 33.8% at 10 μg/mL of DTPP after irradiation with 2.4 J/cm2. Oxidative stress was produced by ROS in PDT and non-reversible cell oxidative damage appeared due to the cells’ modulation of the oxidative stress balance during the PDT response. Chr had a- effect on ROS capture and an inhibitory effect on the PDT-induced destruction of cell membranes. ConclusionsSH-SY5Y cells were susceptible to DTPP-PDT, resulting in a mitochondrial apoptosis pathway. There is an antagonistic effect of Chr on PDT in SH-SY5Y neuroblastoma cells.

Commentary: Photodynamic Therapy-Induced Oxidative Stress and the Circadian Rhythm

Commentary: Photodynamic Therapy-Induced Oxidative Stress and the Circadian Rhythm

Revealing the Photodynamic Stress In Situ with a Dual-Mode Two-Photon 1O2 Fluorescent Probe.

Singlet oxygen (1O2) plays significant physiological and pathological functions, especially in causing photodynamic stress in vivo. However, specific 1O2 monitoring is an immense challenge, owing to its short half-lives and high oxidizing ability. To address this, we engineered three photostable two-photon fluorescence probe NBs for highly efficient 1O2 monitoring based on bioinspired novel tryptophan derivatives, among which NB-MOT was the best one comprehensively. Upon being cracked with 1O2, NB-MOT rapidly (within 5 s) demonstrated a remarkable enhancement in fluorescence intensity (∼180 fold) and lifetime (∼18 fold). Taking these advantages into account, NB-MOT was applied to evaluate exogenous and endogenous 1O2 in diverse biosystems. We successfully tracked the intracellular 1O2 level during photodynamic therapy, and for the first time achieved 1O2 mapping in live cells with dual-mode imaging as well as revealed ciprofloxacin-induced photodynamic stress in mice. NB-MOT was thus believed to be of instructive significance for studying the 1O2-mediated stress in wider biological milieus.

Altered regulation of porphyrin biosynthesis and protective responses to acifluorfen-induced photodynamic stress in transgenic rice expressing Bradyrhizobium japonicum Fe-chelatase

We examined the molecular regulation of porphyrin biosynthesis and protective responses in transgenic rice (Oryza sativa) expressing Bradyrhizobium japonicum Fe-chelatase (BjFeCh) after treatment with acifluorfen (AF). During the photodynamic stress imposed by AF, transcript levels of BjFeCh in transgenic plants increased greatly; moreover, transcript levels of OsFeCh2 remained almost constant, whereas in wild type (WT) plants they were considerably down-regulated. In the heme branch, transgenic plants exhibited greater levels of OsFC and HO transcripts than WT plants in the untreated stems as well as in the AF-treated leaves and stems. Both WT and transgenic plants treated with AF substantially decreased transcript levels for all the genes in the chlorophyll branch, with less decline in transgenic plants. After AF treatment, ascorbate (Asc) content and the redox Asc state greatly decreased in leaves of WT plants; however, in transgenic plants both parameters remained constant in leaves and the Asc redox state increased by 20% in stems. In response to AF, the leaves of WT plants greatly up-regulated CatA, CatB, and GST compared to those of transgenic plants, whereas, in the stems, transgenic plants showed higher levels of CatA, CatC, APXb, BCH, and VDE. Photochemical quenching, qP, was considerably dropped by 31% and 18% in WT and transgenic plants, respectively in response to AF, whereas non-radiative energy dissipation through non-photochemical quenching increased by 77% and 38% in WT and transgenic plants, respectively. Transgenic plants treated with AF exhibited higher transcript levels of nucleus-encoded photosynthetic genes, Lhcb1 and Lhcb6, as well as levels of Lhcb6 protein compared to those of WT plants. Our study demonstrates that expression of BjFeCh in transgenic plants influences not only the regulation of porphyrin biosynthesis through maintaining higher levels of gene expression in the heme branch, but also the Asc redox function during photodynamic stress caused by AF.

Oxidative stress in neonates with hyperbilirubinemia before and after phototherapy: malondialdehyde and catalase activity

Background Phototherapy is used to treat neonatal hyperbilirubinemia, but is currently thought to cause photodynamic stress and can induce lipid peroxidation. There is increasing evidence that many severe diseases of the neonates are caused by oxidative injury and lipid peroxidation. In the present communique, we review the oxidative succeptibility of the neonate and the evidence now available that phototherapy induces oxidative stress. Malondialdehyde (MDA) is a metabolic product of free radicals. Catalase is a antioxidant that binds free radicals.
 Objective To compare the levels of oxidants and antioxidants before and after phototherapy in neonates with hyperbilirubinemia.
 Methods This pretest-posttest control group study was conducted in Sanglah Hospital, Bali from November 2016 to April 2017. Thirty babies with gestational age ≥35 weeks and hyperbilirubinemia with total bilirubin levels requiring phototherapy were included in this study. The MDA levels and catalase activity were measured before and after 24 hours of phototherapy.
 Results Comparative analysis using paired T-test showed a significant increase of malondialdehyde level, with mean MDA 23.73 (SD 8.20) nmol/mL before and 53.05 (SD 10.18) nmol/mL after phototherapy (P<0.001). However, catalase activity significantly decreased from of 72.33 (SD 10.63) kU/L before phototherapy to 44.85 (SD 14.79) kU/L after phototherapy (P<0.001). The MDA level had a significant, negative association with catalase activity after phototherapy (r =-0.4; P=0.028).
 Conclusion Neonates with hyperbilirubinemia are found to have increased oxidative stress after phototherapy, as indicated by increased MDA levels and decreased CAT activity after 24 hours of phototherapy.

Lipofuscin-mediated photodynamic stress induces adverse changes in nanomechanical properties of retinal pigment epithelium cells

Retinal pigment epithelium (RPE) is an important part of the blood-retina barrier (BRB) that separates the retina from the choroid. Although melanin granules contribute to the mechanical stability of the BRB complex, it is unknown if the age pigment lipofuscin affects mechanical properties of the tissue. To address this issue the effect of sub-lethal photic stress mediated by phagocytized lipofuscin granules, isolated from RPE of human donors, on morphology and mechanical properties of ARPE-19 cells was investigated. Nanomechanical analysis using atomic force spectroscopy revealed that irradiation of cells containing lipofuscin granules with blue light induced significant softening of the cells, which was accompanied by substantial reorganization of the cell cytoskeleton due to peroxidation of cellular proteins. Our results indicate that lipofuscin-mediated photic stress can cause significant modification of the RPE cells with the potential to disturb biological function of the BRB complex.

Radiation-induced bystander and systemic effects serve as a unifying model system for genotoxic stress responses.

This review summarises our current understanding of the radiation-induced bystander/systemic effect (RIBE) as well as other types of intercellular reactions induced by malignant tumours, chemotherapy, photodynamic stress and the microbiome. On the basis of striking similarities between these different types of responses RIBE is proposed as a prototype model of a unifying genotoxic stress response system. The early bystander response is initiated by a mitochondria-dependent increase of reactive oxygen species (ROS) and triggers a complex intercellular signalling cascade leading to a sustained increase of cellular DNA damage. The susceptibility to this DNA damage-inducing signal depends on the genetic make-up of the recipient cell population where ATR/ATM- and FA/BRCA-dependent DNA damage response pathways are key players. Long distance bystander/systemic effects observed in in vivo-models are sustained by macrophage-mediated inflammation. Of clinical importance is the potential contribution of bystander DNA damage to an increased risk of malignancies. Defects in DNA damage repair pathways are frequently observed in tumours, which may affect their susceptibility to bystander DNA damage. The potential role for molecular targeted inhibitors in the therapeutic exploitation of bystander responses as well as their differential modulation of targeted and non-targeted effects is also discussed in this context.

Sublethal Photodynamic Treatment Does Not Lead to Development of Resistance.

A promising new alternative approach for eradication of antibiotic-resistant strains is to expose microbes to photosensitizers, which upon illumination generate reactive oxygen species. Among the requirements for a potent, medically applicable photosensitizer, are high efficacy in killing microbes and low toxicity to the host. Since photodynamic treatment is based on production of reactive species which are potentially DNA damaging and mutagenic, it might be expected that under selective pressure, microbes would develop resistance. The aim of this study was to determine if antibacterial photodynamic treatment with a highly photoefficient photosensitizer, Zn(II) meso-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin would lead to development of resistance. To answer that question, bacterial cultures were subjected to multiple cycles of sublethal photodynamic stress and regrowth, and to continuous growth under photodynamic exposure. Antibiotic-resistant Staphylococcus aureus and Escherichia coli clinical isolates were also tested for susceptibility to photodynamic inactivation and for development of resistance. Results demonstrated that multiple photodynamic exposures and regrowth of surviving cells or continuous growth under sublethal photodynamic conditions, did not lead to development of resistance to photosensitizers or to antibiotics. Antibiotic-resistant E. coli and S. aureus were as sensitive to photodynamic killing as were their antibiotic-sensitive counterparts and no changes in their sensitivity to antibiotics or to photodynamic inactivation after multiple cycles of photodynamic treatment and regrowth were observed. In conclusion, photosensitizers with high photodynamic antimicrobial efficiency can be used successfully for eradication of antibiotic-resistant bacterial strains without causing development of resistance.

Plant Photodynamic Stress: What's New?

In the 1970's, an unconventional stressful photodynamic treatment applied to plants was investigated in two directions. Exogenous photosensitizer treatment underlies direct photodynamic stress while treatment mediating endogenous photosensitizer over-accumulation pinpoints indirect photodynamic stress. For indirect photodynamic treatment, tetrapyrrole biosynthesis pathway was deregulated by 5-aminolevulenic acid or diphenyl ether. Overall, photodynamic stress involves the generation of high amount of reactive oxygen species leading to plant cell death. All these investigations were mainly performed to gain insight into new herbicide development but they were rapidly given up or limited due to the harmfulness of diphenyl ether and the high cost of 5-aminolevulinic acid treatment. Twenty years ago, plant photodynamic stress came back by way of crop transgenesis where for example protoporphyrin oxidases from human or bacteria were overexpressed. Such plants grew without dramatic effects of photodamage suggesting that plants tolerated induced photodynamic stress. In this review, we shed light on the occurrence of plant photodynamic stress and discuss challenging issues in the context of agriculture focusing on direct photodynamic modality. Indeed, we highlighted applications of exogenous PS especially porphyrins on plants, to further develop an emerged antimicrobial photodynamic treatment that could be a new strategy to kill plant pathogens without disturbing plant growth.

Responses of an adventitious fast‐growing plant to photodynamic stress: comparative study of anionic and cationic porphyrin effect on Arabidopsis thaliana

Antimicrobial photodynamic treatment (APDT) based on the use of a photosensitizer to produce reactive oxygen species (ROS) that induce cell death could be envisaged to fight against plant pathogens. For setting this strategy, we want to study how plants themselves respond to photodynamic treatment. In previous work we showed that tomato plantlets were able to resist photoactivated tetra (N-methylpyridyl) porphyrin (CP) or the zinc metalated form (CP-Zn). To enlarge our plant expertise related to exogenous porphyrins treatment and to further defend this approach, we studied how a weed like Arabidopsis thaliana responded to exogenous supply of anionic and cationic porphyrins. Both types of photosensitizers had no negative effect on seed germination and did not hamper the development etiolated Arabidopsis plantlet under dark conditions. Thus, post-emergence effects of porphyrin photoactivation on the development of 14day-old in vitro Arabidopsis plantlet under light were observed. CP-Zn was the most efficient photosensitizer to kill Arabidopsis plantlets while anionic tetra (4-sulfonatophenyl) porphyrin only delayed their growth and development. Indeed only 7% of plantlets could be rescued after CP-Zn treatment. Furthermore, non-enzymatic and enzymatic defense components involved in detoxification of ROS generated by CP-Zn under illumination were downregulated or stable with the exception of sevenfold increase in proline content. As previously demonstrated in the literature for microbial agents and in the present work for Arabidopsis, CP-Zn was efficient enough to eradicate unwanted vegetation and plant pathogens without at the same time killing plants of agronomic interest such as tomato plantlets.

Bystander effects of nitric oxide in anti-tumor photodynamic therapy.

Ionizing radiation of specifically targeted cells in a given population is known to elicit pro-death or pro-survival responses in non-targeted bystander cells, which often make no physical contact with the targeted ones. We have recently demonstrated a similar phenomenon for non-ionizing photodynamic therapy (PDT), showing that prostate cancer cells subjected to targeted photodynamic stress stimulated growth and migration of non-stressed, non-contacting bystander cells. Diffusible nitric oxide (NO) generated by stress-upregulated inducible nitric oxide synthase (iNOS) was shown to play a dominant role in these responses. Moreover, target-derived NO stimulated iNOS/NO induction in bystanders, suggesting a NO-mediated feed-forward field effect driven by targeted cells surviving the photodynamic challenge. In this research highlight, we will review these findings and discuss their potential negative implications on clinical PDT outcomes and how these might be mitigated through pharmacologic use of select iNOS inhibitors.

Zeaxanthin and α-tocopherol reduce the inhibitory effects of photodynamic stress on phagocytosis by ARPE-19 cells

Zeaxanthin and α-tocopherol have been previously shown to efficiently protect liposomal membrane lipids against photosensitized peroxidation, and to protect cultured RPE cells against photodynamic killing. Here the protective action of combined zeaxanthin and α-tocopherol was analyzed in ARPE-19 cells subjected to photodynamic (PD) stress mediated by rose Bengal (RB) or merocyanine-540 (MC-540) at sub-lethal levels. Stress-induced cytotoxicity was analyzed by the MTT assay. The peroxidation of membrane lipids was determined by HPLC-EC (Hg) measurements of cholesterol hydroperoxides using cholesterol as a mechanistic reporter molecule. The specific phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was measured by flow cytometry, and the levels of phagocytosis receptor proteins αv integrin subunit, β5 integrin subunit and MerTK were quantified by Western blot analysis. Cytotoxicity measures confirmed that PD stress levels used for phagocytosis analysis were sub-lethal and that antioxidant supplementation protected against higher, lethal PD doses. Sub-lethal PD stress mediated by both photosensitizers induced the accumulation of 5α-OOH and 7α/β-OOH cholesterol hydroperoxides and the addition of the antioxidants substantially inhibited their accumulation. Antioxidant delivery prior to PD stress also reduced the inhibitory effect of stress on POS phagocytosis and partially reduced the stress-induced diminution of phagocytosis receptor proteins. The use of a novel model system where oxidative stress was induced at sub-lethal levels enable observations that would not be detectable using lethal stress models. Moreover, novel observations about the protective effects of zeaxanthin and α-tocopherol on photodynamic damage to ARPE-19 cell membranes and against reductions in the abundance of receptor proteins involved in POS phagocytosis, a process essential for photoreceptor survival, supports the importance of the antioxidants in protecting of the retina against photooxidative injury.

P53 and Ca(2+) signaling from the endoplasmic reticulum: partners in anti-cancer therapies.

Ca(2+) transfer from the endoplasmic reticulum (ER) to the mitochondria critically controls cell survival and cell death decisions. Different oncogenes and deregulation of tumor suppressors exploit this mechanism to favor the survival of altered, malignant cells. Two recent studies of the Pinton team revealed a novel, non-transcriptional function of cytosolic p53 in cell death. During cell stress, p53 is recruited to the ER and the ER-mitochondrial contact sites. This results in augmented ER Ca(2+) levels by enhancing sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) activity, ultimately promoting mitochondrial Ca(2+) overload. The boosting of "toxic" Ca(2+) signaling by p53 appears to be a critical component of the cell death-inducing properties of chemotherapeutic agents and anti-cancer treatments, like photodynamic stress. Strikingly, the resistance of p53-deficient cancer cells to these treatments could be overcome by facilitating Ca(2+) transfer between the ER and the mitochondria via overexpression of SERCA or of the mitochondrial Ca(2+) uniporter (MCU). Importantly, these concepts have also been supported by in vivo Ca(2+) measurements in tumor masses in mice. Collectively, these studies link for the first time the major tumor suppressor, p53, to Ca(2+) signaling in dictating cell-death outcomes and by the success of anti-cancer treatments.

Differential antioxidant defense and detoxification mechanisms in photodynamically stressed rice plants treated with the deregulators of porphyrin biosynthesis, 5-aminolevulinic acid and oxyfluorfen

This study focuses on differential molecular mechanisms of antioxidant and detoxification systems in rice plants under two different types of photodynamic stress imposed by porphyrin deregulators, 5-aminolevulinic acid (ALA) and oxyfluorfen (OF). The ALA-treated plants with white necrosis exhibited a greater decrease in photochemical quantum efficiency, Fv/Fm, as well as a greater increase in activity of superoxide dismutase, compared to the OF-treated plants. By contrast, the brown necrosis in OF-treated plants resulted in not only more widely dispersed H2O2 production and greater increases in H2O2-decomposing enzymes, catalase and peroxidase, but also lower ascorbate redox state. In addition, ALA- and OF-treated plants markedly up-regulated transcript levels of genes involved in detoxification processes including transport and movement, cellular homeostasis, and xenobiotic conjugation, with prominent up-regulation of serine/threonine kinase and chaperone only in ALA-treated plants. Our results demonstrate that different photodynamic stress imposed by ALA and OF developed differential actions of antioxidant enzymes and detoxification. Particularly, detoxification system may play potential roles in plant protection against photodynamic stress imposed by porphyrin deregulators, thereby contributing to alleviation of photodynamic damage.

Perturbed porphyrin biosynthesis contributes to differential herbicidal symptoms in photodynamically stressed rice (Oryza sativa) treated with 5-aminolevulinic acid and oxyfluorfen

This paper focuses on the molecular mechanism of deregulated porphyrin biosynthesis in rice plants under photodynamic stress imposed by an exogenous supply of 5-aminolevulinic acid (ALA) and oxyfluorfen (OF). Plants treated with 5 mM ALA or 50 µM OF exhibited differential herbicidal symptoms as characterized by white and brown necrosis, respectively, with substantial increases in cellular leakage and malondialdehyde production. Protoporphyrin IX accumulated to higher levels after 1 day of ALA and OF treatment, whereas it decreased to the control level after 2 days of ALA treatment. Plants responded to OF by greatly decreasing the levels of Mg–protoporphyrin IX (MgProto IX), MgProto IX methyl ester, and protochlorophyllide to levels lower than control, whereas their levels drastically increased 1 day after ALA treatment and then disappeared 2 days after the treatment. Enzyme activity and transcript levels of HEMA1, GSA and ALAD for ALA synthesis greatly decreased in ALA- and OF-treated plants. Transcript levels of PPO1, CHLH, CHLI, and PORB genes involving Mg–porphyrin synthesis continuously decreased in ALA- and OF-treated plants, with greater decreases in ALA-treated plants. By contrast, up-regulation of FC2 and HO2 genes in Fe–porphyrin branch was noticeable in ALA and OF-treated plants 1 day and 2 days after the treatments, respectively. Decreased transcript levels of nuclear-encoded genes Lhcb1, Lhcb6, and RbcS were accompanied by disappearance of MgProto IX in ALA- and OF-treated plants after 2 days of the treatments. Under photodynamic stress imposed by ALA and OF, tight control of porphyrin biosynthesis prevents accumulation of toxic metabolic intermediates not only by down-regulation of their biosynthesis but also by photodynamic degradation. The up-regulation of FC2 and HO2 also appears to compensate for the photodynamic stress-induced damage.

Spectral and kinetic parameters of phosphorescence of triplet chlorophyll a in the photosynthetic apparatus of plants.

Spectral and kinetic parameters and quantum yield of IR phosphorescence accompanying radiative deactivation of the chlorophyll a (Chl a) triplet state were compared in pigment solutions, greening and mature plant leaves, isolated chloroplasts, and thalluses of macrophytic marine algae. On the early stages of greening just after the Shibata shift, phosphorescence is determined by the bulk Chl a molecules. According to phosphorescence measurement, the quantum yield of triplet state formation is not less than 25%. Further greening leads to a strong decrease in the phosphorescence yield. In mature leaves developing under normal irradiation conditions, the phosphorescence yield declined 1000-fold. This parameter is stable in leaves of different plant species. Three spectral forms of phosphorescence-emitting chlorophyll were revealed in the mature photosynthetic apparatus with the main emission maxima at 955, 975, and 995 nm and lifetimes ~1.9, ~1.5, and 1.1-1.3 ms. In the excitation spectra of chlorophyll phosphorescence measured in thalluses of macrophytic green and red algae, the absorption bands of Chl a and accessory pigments - carotenoids, Chl b, and phycobilins - were observed. These data suggest that phosphorescence is emitted by triplet chlorophyll molecules that are not quenched by carotenoids and correspond to short wavelength forms of Chl a coupled to the normal light harvesting pigment complex. The concentration of the phosphorescence-emitting chlorophyll molecules in chloroplasts and the contribution of these molecules to chlorophyll fluorescence were estimated. Spectral and kinetic parameters of the phosphorescence corresponding to the long wavelength fluorescence band at 737 nm were evaluated. The data indicate that phosphorescence provides unique information on the photophysics of pigment molecules, molecular organization of the photosynthetic apparatus, and mechanisms and efficiency of photodynamic stress in plants.

Nitrosylhemoglobin in photodynamically stressed human tumors growing in nude mice

The role of nitric oxide in human tumor biology and therapy has been the subject of extensive studies. However, there is only limited knowledge about the mechanisms of NO production and its metabolism, and about the role NO can play in modern therapeutic procedures, such as photodynamic therapy. Here, for the first time, we report the presence of nitrosylhemoglobin, a stable complex of NO, in human lung adenocarcinoma A549 tumors growing in situ in nude mice. Using electron paramagnetic resonance spectroscopy we show that the level of nitrosylhemoglobin increases in the course of photodynamic therapy and that the phenomenon is local. Even the destruction of strongly vascularized normal liver tissue did not induce the paramagnetic signal, despite bringing about tissue necrosis. We conclude that photodynamic stress substantiates NO production and blood extravasation in situ, both processes on-going even in non-treated tumors, although at a lower intensity.

Bacteriophytochrome controls carotenoid-independent response to photodynamic stress in a non-photosynthetic rhizobacterium, Azospirillum brasilense Sp7

Ever since the discovery of the role of bacteriophytochrome (BphP) in inducing carotenoid synthesis in Deinococcus radiodurans in response to light the role of BphPs in other non-photosynthetic bacteria is not clear yet. Azospirillum brasilense, a non-photosynthetic rhizobacterium, harbours a pair of BphPs out of which AbBphP1 is a homolog of AtBphP1 of Agrobacterium tumefaciens. By overexpression, purification, biochemical and spectral characterization we have shown that AbBphP1 is a photochromic bacteriophytochrome. Phenotypic study of the ΔAbBphP1 mutant showed that it is required for the survival of A. brasilense on minimal medium under red light. The mutant also showed reduced chemotaxis towards dicarboxylates and increased sensitivity to the photooxidative stress. Unlike D. radiodurans, AbBphP1 was not involved in controlling carotenoid synthesis. Proteome analysis of the ΔAbBphP1 indicated that AbBphP1 is involved in inducing a cellular response that enables A. brasilense in regenerating proteins that might be damaged due to photodynamic stress.