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Histochemical and immunohistochemical analysis of enzymes involved in phenolic metabolism during berry development in Vitis vinifera L.

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Abstract

Phenolics are involved in many of plants’ biological functions. In particular, they play important roles in determining the quality of grape berries and the wine made from them, and can also act as antioxidants with beneficial effects for human health. Several enzymes are involved in the synthesis of phenolic compounds. Among them, stilbene synthase (STS) is a key to the biosynthesis of stilbenes, which are considered to be important secondary metabolites in plants. Other enzymes, such as polyphenol oxidase (PPO) and peroxidase (POD), are involved in the degradation of phenolics, and become activated during late stages of berry ripening. In the present study, Vitis vinifera L. berries were sampled at eight stages of development, from 10 days after anthesis to late harvest. The PPO and POD enzymatic activities were determined at each stage. The presence of STS, PPO, and POD proteins in the grape exocarp and mesocarp was detected immunohistochemically and histochemically. The amount and intensity of the immunohistochemical and histochemical signals correlate with the variations in enzyme activities throughout fruit development. Strong STS immunoreactivity was detected until the onset of ripening. Labeled tissue increased gradually from mesocarp to exocarp, showing an intense signal in epidermis. At subcellular level, STS was mainly detected in cytoplasm grains and cell walls. The amount of PPO immunoreactivity increased progressively until the end of ripening. The PPO signal was detected in hypodermal layers and, to a lesser extent, in mesocarp parenchyma cells, especially in cytoplasm grains and cell walls. Finally, POD activity was stronger at the onset of ripening, and the POD histochemical signal was mainly detected in the cell walls of both exocarp and mesocarp tissue.

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Acknowledgements

During this work, M.E.M.C was a recipient of a PhD studentship from the Junta de Extremadura (PD12106). We also greatly thank Dr. Roque Bru (Departamento de Agroquímica y Bioquímica, University of Alicante, Spain) for the generous gifts of rabbit anti-PPO polyclonal antibody.

Funding

This work was supported by grants from Junta de Extremadura (GR15158 and AGA001), Fondo Social Europeo and Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA-FEDER RTA-2012-00029-C01).

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  1. María Eugenia Molero de Ávila and María Victoria Alarcón contributed equally to this work.

Authors and Affiliations

  1. Área de Biología Celular, Departamento de Anatomía, Biología Celular y Zoología, Facultad de Ciencias, Universidad de Extremadura, Avda. de Elvas s/n, 06006, Badajoz, Spain

    María Eugenia Molero de Ávila & Javier Francisco-Morcillo

  2. Departamento de Hortofruticultura, Instituto de Investigaciones Agrarias Finca “La Orden-Valdesequera”, CICYTEX, Junta de Extremadura, Guadajira, 06187, Badajoz, Spain

    María Victoria Alarcón, David Uriarte & Luis Alberto Mancha

  3. Departamento de Enología, INTAEX, CICYTEX, Junta de Extremadura, 06071, Badajoz, Spain

    Daniel Moreno

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  1. María Eugenia Molero de Ávila
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  2. María Victoria Alarcón
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Correspondence to Javier Francisco-Morcillo.

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Molero de Ávila, M.E., Alarcón, M.V., Uriarte, D. et al. Histochemical and immunohistochemical analysis of enzymes involved in phenolic metabolism during berry development in Vitis vinifera L.. Protoplasma 256, 25–38 (2019). https://doi.org/10.1007/s00709-018-1278-1

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