Multiple origins of green coloration in frogs mediated by a novel biliverdin-binding serpin.

Many vertebrates have distinctive blue-green bones and other tissues due to unusually high biliverdin concentrations-a phenomenon called chlorosis. Despite its prevalence, the biochemical basis, biology, and evolution of chlorosis are poorly understood. In this study, we show that the occurrence of high biliverdin in anurans (frogs and toads) has evolved multiple times during their evolutionary history, and relies on the same mechanism-the presence of a class of serpin family proteins that bind biliverdin. Using a diverse combination of techniques, we purified these serpins from several species of nonmodel treefrogs and developed a pipeline that allowed us to assemble their complete amino acid and nucleotide sequences. The described proteins, hereafter named biliverdin-binding serpins (BBS), have absorption spectra that mimic those of phytochromes and bacteriophytochromes. Our models showed that physiological concentration of BBSs fine-tune the color of the animals, providing the physiological basis for crypsis in green foliage even under near-infrared light. Additionally, we found that these BBSs are most similar to human glycoprotein alpha-1-antitrypsin, but with a remarkable functional diversification. Our results present molecular and functional evidence of recurrent evolution of chlorosis, describe a biliverdin-binding protein in vertebrates, and introduce a function for a member of the serpin superfamily, the largest and most ubiquitous group of protease inhibitors.

Naturally occurring fluorescence in frogs.

Fluorescence, the absorption of short-wavelength electromagnetic radiation reemitted at longer wavelengths, has been suggested to play several biological roles in metazoans. This phenomenon is uncommon in tetrapods, being restricted mostly to parrots and marine turtles. We report fluorescence in amphibians, in the tree frog Hypsiboas punctatus, showing that fluorescence in living frogs is produced by a combination of lymph and glandular emission, with pigmentary cell filtering in the skin. The chemical origin of fluorescence was traced to a class of fluorescent compounds derived from dihydroisoquinolinone, here named hyloins. We show that fluorescence contributes 18-29% of the total emerging light under twilight and nocturnal scenarios, largely enhancing brightness of the individuals and matching the sensitivity of night vision in amphibians. These results introduce an unprecedented source of pigmentation in amphibians and highlight the potential relevance of fluorescence in visual perception in terrestrial environments.

Effect of arsenic on reflectance spectra and chlorophyll fluorescence of aquatic plants.

Arsenic pollution of groundwater is a serious problem in many regions of Latin America that causes severe risks to human health. As a consequence, non-destructive monitoring methodologies, sensitive to arsenic presence in the environment and able to perform a rapid screening of large polluted areas, are highly sought-after. Both chlorophyll - a fluorescence and reflectance of aquatic plants may be potential indicators to sense toxicity in water media. In this work, the effects of arsenic on the optical and photophysical properties of leaves of different aquatic plants (Vallisneria gigantea, Azolla filiculoides and Lemna minor) were evaluated. Reflectance spectra were recorded for the plant leaves from 300 to 2400 nm. The spectral distribution of the fluorescence was also studied and corrected for light re-absorption processes. Photosynthetic parameters (Fv/Fm and ΦPSII) were additionally calculated from the variable chlorophyll fluorescence recorded with a pulse amplitude modulated fluorometer. Fluorescence and reflectance properties for V. gigantea and A. filiculoides were sensitive to arsenic presence in contrast to the behaviour of L. minor. Observed changes in fluorescence spectra could be interpreted in terms of preferential damage in photosystem II. The quantum efficiency of photosystem II for the first two species was also affected, decreasing upon arsenic treatment. As a result of this research, V. gigantea and A. filiculoides were proposed as bioindicators of arsenic occurrence in aquatic media.

Atrazine and Methyl Viologen Effects on Chlorophyll-a Fluorescence Revisited-Implications in Photosystems Emission and Ecotoxicity Assessment.

In this work, we use the effect of herbicides that affect the photosynthetic chain at defined sites in the photosynthetic reaction steps to derive information about the fluorescence emission of photosystems. The interpretation of spectral data from treated and control plants, after correction for light reabsorption processes, allowed us to elucidate current controversies in the subject. Results were compatible with the fact that a nonnegligible Photosystem I contribution to chlorophyll fluorescence in plants at room temperature does exist. In another aspect, variable and nonvariable chlorophyll fluorescence were comparatively tested as bioindicators for detection of both herbicides in aquatic environment. Both methodologies were appropriate tools for this purpose. However, they showed better sensitivity for pollutants disconnecting Photosystem II-Photosystem I by blocking the electron transport between them as Atrazine. Specifically, changes in the (experimental and corrected by light reabsorption) red to far red fluorescence ratio, in the maximum photochemical quantum yield and in the quantum efficiency of Photosytem II for increasing concentrations of herbicides have been measured and compared. The most sensitive bioindicator for both herbicides was the quantum efficiency of Photosystem II.

Optical properties of the adaxial and abaxial faces of leaves. Chlorophyll fluorescence, absorption and scattering coefficients.

Emission fluorescence spectra were obtained for the adaxial and abaxial faces of dicotyledonous (Ficus benjamina L., Ficus elastica, Gardenia jasminoides and Hedera helix) and monocotyledonous leaves (Gladiolus spp. and Dracaena cincta bicolor). After correction by light-re-absorption processes, using a previously published physical model, the adaxial faces of dicotyledons showed a fluorescence ratio Fred/Ffar-red rather lower than the respective values for the abaxial faces. Monocotyledons and shade-adapted-plants showed similar values for the corrected fluorescence ratio for both faces. Even when differences in experimental fluorescence emission from adaxial and abaxial leaves in dicotyledons are mostly due to light re-absorption processes, the residual dissimilarity found after application of the correction model would point to the fact that fluorescence re-absorption is not the only responsible for the observed disparity. It was concluded that light re-absorption processes does not account entirely for the differences in the experimental emission spectra between adaxial and abaxial leaves. Differences that remains still present after correction might be interpreted in terms of a different photosystem ratio (PSII/PSI). Experiments at low temperature sustained this hypothesis. In dicotyledons, light reflectance for adaxial leaves was found to be lower than for the abaxial ones. It was mainly due to an increase in the scattering coefficient for the lower leaf-side. The absorption coefficient values were slightly higher for the upper leaf-side. During senescence of Ficus benjamina leaves, the scattering coefficient increased for both the upper and lower leaf-sides. With senescence time the absorption coefficient spectra broadened while the corrected fluorescence ratio (Fred/Ffar-red) decreased for both faces. The results pointed to a preferential destruction of photosystem II relative to photosystem I during senescence.

Re-absorption of chlorophyll fluorescence in leaves revisited. A comparison of correction models.

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.

A model considering light reabsorption processes to correct in vivo chlorophyll fluorescence spectra in apples.

Chlorophyll-a contained in the peel of Granny Smith apples emits fluorescence upon excitation with blue light. The observed emission, collected by an external detector and corrected by its spectral response, is still distorted by light reabsorption processes taking place in the fruit skin and differs appreciably from the true spectral distribution of fluorescence emerging from chlorophyll molecules in the biological tissue. Reabsorption processes particularly affect the ratio of fluorescence intensities at 680 nm and at 730 nm. A model to obtain the correct spectral distribution of the emission, from the experimental fluorescence recorded at a fluorometer detector and corrected for the detector spectral sensitivity, is developed in the present work. Measurements of the whole fruit reflectance, the peel transmittance and the flesh reflectance allow the calculation of the reabsorption-corrected spectra. The model is validated by comparing the corrected emission spectra with that obtained for a thin layer of apple-peel-chloroplasts, where no reabsorption takes place. It is recommended to correct distortions in emission spectra of intact fruits due to light reabsorption effects whenever a correlation between the physiological state of the fruit and its fluorescence spectra is investigated.