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In situ fluorescence measurements have been used to investigate relative amounts of blue-green pigments and their distributions in plant leaves from Euphorbia pulcherrima. Advantage was taken from the fact that this species has white leaves on the top, with low pigment concentrations, and green leaves on the stem with ordinary pigment concentrations. Excitation- and emission spectra below 410 nm from white leaves, where pigment absorption is low, are not distorted by self-absorption. Absorption- and reflection spectra from white and green leaves were measured using a spectrophotometer equipped with an integrating sphere. The absorption spectra were used to correct recorded fluorescence spectra for self-absorption. Self-absorption corrected photosystem fluorescence from green leaves, modeling light transmission in leaf tissue exponentially, matches to the excitation/emission spectra from white leaves, apart from small differences due to the pigment concentrations and selective scattering. The introduced exponentially decaying transmission relation also predicts that the ratio of excitation spectra from a white and green leaf is in proportion to the absorption spectrum of the green leaf, which was validated for Photosystem II particle fluorescence. This relation was also used to find a scaled absorption spectrum responsible for blue-green emission, which was assumed to originate from lignin. Excitation/emission spectra of the blue-green fluorescence were decomposed into five components and their relative amounts from adaxial and abaxial sides of the leaves have been quantified. Fluorescence lifetime measurements of the leaves, upon 403 nm excitation, revealed three decay times corresponding to the lignin fluorophores emitting in blue and green spectral region, and indicated that emissions at 500 and 550 nm may originate from the same fluorophore residing in the two physically different environments.

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Laser excitation of a single precursor, namely 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (HHEMP), has been used for generating the radical cations and radical anions of various carotenoids in methanol. In the presence of oxygen, laser excitation of HHEMP undergoes an efficient α-cleavage reaction (Norrish type I) to form acyl radicals, which react with O2, in a nearly diffusion-controlled reaction, to form their corresponding strong oxidizing acylperoxyl radicals (RO2•) (E = ~1.1 V (v SHE)), which are capable of oxidizing almost all carotenoids. Under argon-saturated conditions and in the presence of strong base (0.01 M NaOH or tetrabutylammonium hydroxide (TBAOH)), the initially formed 2-hydroxy-2-propyl radical (ACH•), generated after LFP of HHEMP, is deprotonated to form the strong reducing acetone ketyl radical (AC•-) (E {acetone/ AC•-} = -2.1 V (v SHE)), which is capable of reducing all carbonyl-containing carotenoids. To validate this new proposed approach, retinal and ß-apo-8'-carotenal (APO), with known spectroscopic data, were investigated in methanol, acetonitrile and tetrahydrofuran (THF). In addition, the radical ions of newly investigated carotenoids, namely 4-oxo-ß-apo-15'-carotenoic acid (4-oxo-15'), crocetindial, 4-oxo-ß-apo-10'-carotenoic acid ethyl ester (4-oxo-10') and 4-oxo-ß-apo-8'-carotenoic acid ethyl ester (4-oxo-8') have been reported. Moreover, the scope of this approach has been extended to investigate the radical ions of chlorophyll b.

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For investigations of ongoing processes in plants, such as photosynthesis in conifer leaves, nondestructive and noninvasive measuring techniques are needed. In this paper, a novel approach has been developed for the measurement of chloroplasts' numbers and pigment contents in conifer leaves based on the measurements of leaf absorption spectra using optical fibers and an array spectrophotometer. To eliminate the effect of scattering on the measured absorption spectra, a strategy has been applied taking advantage of the combined use of thin optical fibers normal to the needle's longitudinal axis and the phenomenon that scattering is largest in the forward direction. The optical path in the leaf is nearly the distance between the fiber tips; thus, we were able to obtain the absorption spectrum of the pigments in situ. A effect of the measured absorption spectra, occurring due to the organization of pigments in the leaf and interaction between light and leaf interior, can be accounted for by using the so-called Duysens transformation. Using this transformation, pigment contents and the relative number of chloroplasts can be obtained from the measured absorption spectra. We applied the method to observe pigment concentrations in different stages of the greening process in the leaves of two conifer species, Taxus baccata and Picea abies. The presented method may be used to estimate changes in chloroplast number and pigment content during various phases of greening of a species and to observe differences among various species.

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Vitamin A (retinol) and various natural retinoids are essential for life. Under oxidative conditions, vitamin A radical cation (RET+) can be formed. Many deleterious effects were reported about the formation of carotenoid radical cations in biological environments, on the other hand, little is known about the consequences of the RET+ formation in these environments. Therefore, it is important to explore the reactivity of RET+ toward various biological substrates. Here, we employed nanosecond laser flash photolysis (LFP) to generate RET+ (λmax=580nm in methanol) and examine its reactivity toward a wide range of biological molecules including amino acids, vitamins, carotenoids, naturally-occurring phenols, neurotransmitters such as catecholamines, wide range of phenol derivatives and some selected electron-donors. The results show that the reactivity of RET+ toward various substrates is strongly dependent on the polarity of solvent. In addition, RET+ is able to oxidize amino acids, which subsequently can lead to protein damage. However, the presence of vitamins (vitamins E and C), carotenoids and naturally-occurring phenols (e.g. resveratrol, vanillin, dopamine hydrochloride and l-Dopa) can inhibit the damaging effect of retinol+ by reducing it back to retinol. Vitamin E and carotenoids are the most efficient quenchers for the RET+ (diffusion-controlled reactions). Importantly, our results clearly indicate that the reactivity of RET+ is as strong as that of the powerful trichloromethylperoxyl radical (CCl3O2). Thereby, formation of RET+ in biological media is expected to induce bio-damage.

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Currently there are limitations to gelation strategies to form ionically crosslinked hydrogels, derived in particular from a lack of control over the release kinetics of crosslinking ions, which severely restrict applications. To address this challenge, we describe a new approach to form hydrogels of ionotropic polymers using competitive displacement of chelated ions, thus making specific ions available to induce interactions between polymer chains and form a hydrogel. This strategy enables control of ion release kinetics within an aqueous polymer solution and thus control over gelation kinetics across a wide range of pH. The described technique simplifies or facilitates the use of ionotropic hydrogels in a range of applications, such as 3D printing, microfluidic-based cell encapsulation, injectable preparations and large scale bubble and solid free mouldable gels. We investigate a range of chelator-ion combinations and demonstrate this powerful method to form hydrogels across a wide range of pH and µm-cm length scales. We highlight our findings by applying this gelation strategy to some of the more challenging hydrogel application areas using alginate and polygalacturonate as model polymer systems.

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Chlorosomes are light-harvesting complexes of green photosynthetic bacteria. Chlorosomes contain bacteriochlorophyll (BChl) c, d, or e aggregates that exhibit strong excitonic coupling. The short-range order, which is responsible for the coupling, has been proposed to be augmented by pigment arrangement into undulated lamellar structures with spacing between 2 and 3 nm. Treatment of chlorosomes with hexanol reversibly converts the aggregated chlorosome chlorophylls into a form with spectral properties very similar to that of the monomer. Although this transition has been extensively studied, the structural basis remains unclear due to variability in the obtained morphologies. Here we investigated hexanol-induced structural changes in the lamellar organization of BChl c in chlorosomes from Chlorobium tepidum by a combination of X-ray scattering, electron cryomicroscopy, and optical spectroscopy. At a low hexanol/pigment ratio, the lamellae persisted in the presence of hexanol while the short-range order and exciton interactions between chlorin rings were effectively eliminated, producing a monomer-like absorption. The result suggested that hexanol hydroxyls solvated the chlorin rings while the aliphatic tail partitioned into the hydrophobic part of the lamellar structure. This partitioning extended the chlorosome along its long axis. Further increase of the hexanol/pigment ratio produced round pigment-hexanol droplets, which lost all lamellar order. After hexanol removal the spectral properties were restored. In the samples treated under the high hexanol/pigment ratio, lamellae reassembled in small domains after hexanol removal while the shape and long-range order were irreversibly lost. Thus, all the interactions required for establishing the short-range order by self-assembly are provided by BChl c molecules alone. However, the long-range order and overall shape are imposed by an external structure, e.g., the proteinaceous chlorosome baseplate.

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