Di Meo, F.

Failla, O.

Rocchi, L.

Rustioni, L.

Trouillas, P.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 54 (Special Issue), 173–174 (2015)optical studies of pigment characterization developed by our research team over the past years.Material and Methods: Pigment optical properties have been studied by theoretical chemistry and reflectance spectroscopy. Anthocyanin absorption was described by quantum chemistry calculations based on time-dependent density functional theory (TD-DFT). Further methodologi-cal details are reported in RUSTIONI et al. 2013a. Aiming at a grape color classification, berries were studied by a Lamb-da 35 UV-vis spectrophotometer (Perkin Elmer, Waltham, Massachusetts), completed by a Spectralon integrating sphere (as described in RUSTIONI et al. 2013b). To eluci-date the sunburn physiology with the purpose of resistant cultivar selection, chlorophylls and melanin-like pigments were studied by a Jaz System spectrometer (Ocean Optics) (RUSTIONI et al. 2014).Results and Discussion: The Figure shows the opti-cal properties of grape pigments (anthocyanins), as studied by different methods. These pigments exhibit specific ab-sorption bands in the visible range. Quantum calculations enable in-depth understanding of the electronic origin of these bands, based on the description of excited states from electronic transitions involving π-type molecular orbitals (Figure a). The choice of theoretical methods is critical and requires benchmarking on small and well-documented pro-totypes. An example of grape anthocyanins quantum study is reported in RUSTIONI et al. 2013a. From an experimen-tal view, the absorbance spectra of malvidin-3-O-gluco-side can be obtained after separation by HPLC (Figure b). Nevertheless, to be more consistent with grape molecular composition, we should consider that the berry skin is con-stituted by a mixture of anthocyanins (Figure c), in which malvidin-3-O-glucoside is the major constituent. Compar-ing Figure a, b and c, it is possible to observe slight differ-ences of the maximum absorption wavelengths and of the shapes of the spectra, as evaluated by the different meth-ods. These methods are relatively system- and environ-ment- dependent e.g., the optical properties of the extract-ed anthocyanins are measured in extremely acid solvents able to stabilize the red flavylium cations. The reflectance allows a more adapted description of the actual fruit color, but the obtained spectra (Figure d) should be clean and ac-curate enough. Despite the complexity of grape pigment distribution (high anthocyanin concentration as observed by absorption saturation and presence of other pigment-ed molecules), a reflectance index of pigment concentra-tion has been proposed by our group. Moreover, the berry colors were gathered based on reflectance spectra, produc-ing an objective method in cultivar classification (RUSTIONI et al. 2013b).From the reflectance evaluation, other pigments were also studied, aiming at a cultivar sunburn susceptibil-ity evaluation. In this case, our attention was focused on the photo-oxidative role of chlorophylls and carotenoids. Some reflectance indexes already published were tested and others were developed to quantify these molecules (ROCCHI et al. unpubl.). Melanin-like pigments, the brown phenolic oxidation products yielded under radiative stress, Research NoteAdvances in grape phenotyping: pigment characterization by reflectance and theoretical chemistryL. RUSTIONI1, L. ROCCHI1, F. DI MEO2, O. FAILLA1 and P. TROUILLAS3,41) Università degli Studi di Milano, CIRIVE – Centro Interdipar-timentale di ricerca per l’innovazione in Viticoltura ed Enologia, Milano, Italy2) Division of Theoretical Chemistry, Department of Physics, Chemistry and Biology (IFM), Linköping University, Sweden3) INSERM UMR 850, Univ. Limoges, France4) RCTPM, Palacký University of Olomouc, Czech RepublicK e y  w o r d s :  grape color; optical properties; anthocy-anins; chlorophylls; melanin-like pigments.Introduction: Despite the significant developments over the last decades in molecular biology through the huge production of genetic data, phenotypic records are missing. To complete the biological understanding of the Vitis vinifera L. genetic code expression, a thorough phe-notypic description is mandatory. Due to the variability of the trait natures (e.g. phenology, chemical composition, plant morphology), phenotyping requires the development of specific methodologies. These methods also ideally need to be fast and high-throughput, namely data collection in a short time (following plant phenology) and possibility of replication (in relation to the natural variability particularly due to environmental effects and genetic per environmen-tal (GxE) interactions).Over the past years, our research group has decided to focus on non-invasive methods based on pigment op-tical properties. The color of individual molecules results from the absorption in a specific spectral range and related reflection, and transmission phenomena at these specific wavelengths. As an example, thanks to the π-delocaliza-tion over the entire flavylium cation of anthocyanins, green light is absorbed, resulting in red color (ALLEN 1998). From the optical property rationalization of pigments, it is pos-sible to dissect the reflectance spectra obtained by non-in-vasive measurements, recognizing the absorption bands of the different molecules. In this way, it will be possible to develop algorithms able to describe the chemical composi-tion of fruits. In the framework of the COST Action FA1003 - Grap-enet: East-West Collaboration for Grapevine Diversity Ex-ploration and Mobilization of Adaptive Traits for Breeding - our group has been particularly involved in the phenotyp-ing research area. This note is a synthetic review of the Correspondence to: Dr. L. RUSTIONI, CIRIVE – Centro Inter-dipartimentale di ricerca per l’innovazione in Viticoltura ed Enologia, via G. Celoria 2, I-20133 Milano, Italy. E-mail: laura.rustioni@unimi.it 174 L. RUSTIONI et al.are particularly difficult to be measured due to their struc-tural heterogeneity as well as difficulties in their extrac-tion. The reflectance approach has allowed us the study of these polymers, developing a specific index. In this way, the sunburn symptoms can be objectively recognized and quantified.Conclusions: The understanding of optical properties of grape pigments has allowed paving the way for the de-velopment of high-throughput non-invasive phenotyping methods. The main limitation of this approach is related to the difficult data elaboration, however the speed of data recording (typical of reflectance approach) is particularly adapted to analyze high number of cultivars during the short phenological phase of interest. Joint publication of the COST Action FA1003 "East-West Collaboration for Grapevine Diversity Exploration and Mobiliza-tion of Adaptive Traits for Breeding".ALLEN, M.; 1998: Phenolics demystified. In: Proceedings ASVO Oenol-ogy Seminar. Phenolics and Extraction. Australian Society of Viti-culture and Oenology: Adelaide.ROCCHI, L.; RUSTIONI L.; FAILLA, O.; unpubl.: Chlorophylls and caroten-oids quantification in white grape (Vitis vinifera L.) skins by reflect-ance spectroscopy.RUSTIONI, L.; BASILICO, R.; FIORI, S.; LEONI, S.; LEONI, A.; MAGHRADZE, D.; FAILLA, O.; 2013b: Grape colour phenotyping: development of a method based on the reflectance spectrum. Phytochem. Anal. 24, 453-459.RUSTIONI, L.; DI MEO F.; GUILLAUME, M.; FAILLA, O.; Trouillas P.; 2013a: Tuning color variation in grape anthocyanins at the molecular scale. Food Chem. 141, 4349-4357.RUSTIONI, L.; ROCCHI, L.; GUFFANTI, E.; COLA, G.; FAILLA, O.; 2014: Char-acterization of grape (Vitis vinifera L.) berry sunburn symptoms by reflectance. J. Agric. Food Chem. 62, 3043-3046.Figure: Optical properties of grape anthocyanins studied by dif-ferent methods. a) Theoretical malvidin-3-O-glucoside spectra (the oscillator strength is directly correlated to the absorbance): the excited states are reported in gray and the black line indicates the obtained Gaussian function. b) Absorption spectra of mal-vidin-3-O-glucoside recorded after HPLC separation. c) Grape extract absorbance spectra measured by spectrophotometer. d) Reflectance spectra of red grape skin.

Advances in grape phenotyping: pigment characterization by reflectance and theoretical chemistry

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Advances in grape phenotyping: pigment characterization by reflectance and theoretical chemistry

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