Abstract
The contamination of soils and water with copper (Cu) can compromise the crops production and quality. Fungicides containing Cu are widely and intensively used in viticulture contributing to environmental contamination and genotoxicity in Vitis vinifera L. Despite the difficulty in reproducing field conditions in the laboratory, hydroponic solutions enriched with Cu (1, 10, 25 and 50 μM) were used in forced V. vinifera cuttings to evaluate the DNA damage in leaves of four wine-producing varieties (‘Tinta Barroca’, ‘Tinto Cão’, ‘Malvasia Fina’ and ‘Viosinho’). Alkaline comet assay followed by fluorescence in situ hybridisation (Comet-FISH) was performed with the 45S ribosomal DNA (rDNA) and telomeric [(TTTAGGG)n] sequences as probes. This study aimed to evaluate the tolerance of the four varieties to different concentrations of Cu and to determine which genomic regions were more prone to DNA damage. The comet assay revealed comets of categories 0 to 4 in all varieties. The DNA damage increased significantly (p < 0.001) with the Cu concentration. ‘Tinto Cão’ appeared to be the most sensitive variety because it had the highest DNA damage increase in 50 μM Cu relative to the control. Comet-FISH was only performed on slides of the control and 50 μM Cu treatments. Comets of all varieties treated with 50 μM Cu showed rDNA hybridisation on the head, ‘halo’ and tail (category III), and their frequency was significantly higher than that of control. The frequency of category III comets hybridised with the telomeric probe was only significantly different from the control in ‘Malvasia Fina’ and ‘Tinta Barroca’. Comet-FISH revealed partial damage on rDNA and telomeric DNA in response to Cu but also in control, confirming the high sensitivity of these genomic regions to DNA fragmentation.
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Acknowledgements
The author CC thanks for her grant BI/INTERACT/VW/183/2016 supported by the project INTERACT (‘Integrative Research in Environment, Agro-Chains and Technology’), research line ‘VitalityWine’ (‘Fostering viticulture sustainability for Douro Valley: multidisciplinary efforts from field to wine’), NORTE-01-0145-FEDER-000017, co-financed by the European Regional Development Fund (FEDER) through the North Regional Operational Program 2014/2020 (NORTE 2020). The author AC thanks for her grant BPD/UTAD/INNOVINE&WINE/593/2016 supported by the Project INNOVINE&WINE (‘Vine and Wine Innovation Platform’), research line ‘Viticulture’ (NORTE-01-0145-FEDER-000038, co-financed by FEDER through NORTE 2020), and also thanks to the ‘Fundação para a Ciência e a Tecnologia’ (FCT) and UTAD for her contract as a researcher under the scope of the D.L. n°. 57/2016 of 29 August and Law n.° 57/2017 of 19 July. The authors AC and JL-B thank the COST ACTION ‘INDEPTH’ (CA16212). The authors thank FCT for the projects UIDB/04033/2020 (CITAB/UTAD), UIDB/04046/2020 and UIDP/04046/2020 (BioISI) and UIDB/CVT/00772/2020 (CECAV/UTAD) and also to the Centre of Excellence of Vine and Wine-Regia Douro Park for the availability of the growth chamber.
Funding
This work was supported by the North Regional Operational Program 2014/2020 (NORTE 2020) and co-financed by the European Regional Development Fund (FEDER) by the project INTERACT - (Integrative Research in Environment, Agro-Chains and Technology (NORTE-01-0145-FEDER-000017), Fostering viticulture sustainability for Douro Valley: multidisciplinary efforts from field to wine, research line VitalityWine, and project INNOVINE&WINE (Vine and Wine Innovation Platform, NORTE-01-0145-FEDER-000038, research line Viticulture) and by the grants BI/INTERACT/VW/183/2016 (attributed to author CC) and BPD/UTAD/INNOVINE&WINE/593/2016 (attributed to author AC).
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CC performed the methodology, analysed the data and write the original draft. AC and JL-B were involved in the work conceptualisation, results analyses, writing and review of the manuscript. IG contributed for the methodology optimisation, writing and review of the manuscript.
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Castro, C., Carvalho, A., Gaivão, I. et al. Evaluation of copper-induced DNA damage in Vitis vinifera L. using Comet-FISH. Environ Sci Pollut Res 28, 6600–6610 (2021). https://doi.org/10.1007/s11356-020-10995-7
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DOI: https://doi.org/10.1007/s11356-020-10995-7