Multidisciplinary study of wine-producing grapevine varieties treated with hydroponic solutions enriched with Zinc and Copper
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2021-06-23
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Os solos agrícolas possuem na sua composição vários metais pesados cuja concentração difere entre regiões devido a diferentes propriedades físico-químicas. Porém, a aplicação de agro-químicos à base de metais pesados como o Zinco (Zn) e Cobre (Cu) tem levado à contaminação dos solos agrícolas, em particular os de vinha. Estes metais pesados são também micronutrientes essenciais para as atividades metabólicas das plantas, mas em concentrações reduzidas. A contaminação de solos de vinha com Zn e Cu é ainda mais exacerbada em solo com pH ácido (predominante no Norte de Portugal), uma vez que aumenta a biodisponibilidade de Zn no solo e a translocação do Cu para as plantas, induzindo cito-, geno- e fitotoxicidade. Considerando a importância económica do sector vitivinícola em Portugal e a relevância e o reconhecimento internacional dos vinhos produzidos na região do Douro, esta tese focouse na análise citogenómica e morfo-histológica em raízes e/ou folhas de castas tintas e brancas cultivadas nesta região. Dada a dificuldade em aceder às raízes de videira em vinha, as estacas colhidas foram forçadas e mantidas em ensaios hidropónicos. Estacas das castas Donzelinho Branco, Verdelho e Bastardo foram utilizadas num ensaio hidropónico com solução nutritiva enriquecida com diferentes concentrações de Zn (1.5, 3 e 5 mM). Estacas das castas Tinta Barroca, Tinto Cão, Malvasia Fina e Viosinho foram utilizadas num segundo ensaio hidropónico com solução nutritiva enriquecida com diferentes concentrações de Cu (1, 10, 25 e 50 µM). Estas concentrações de Zn e Cu foram determinadas com base em valores previamente determinados para os solos do Douro ou estabelecidos pela União Europeia. Folhas e raízes recém-formadas durante os ensaios hidropónicos foram utilizadas na otimização de técnicas citogenómicas que foram aplicadas pela primeira vez em Vitis vinifera L. mas que se revelaram úteis na análise de tolerância a diferentes stresses abióticos noutras espécies vegetais. Com este estudo pretendeu-se integrar dados citogenómicos com os obtidos através das análises morfo-histológicas em raízes e/ou folhas no sentido de determinar quais as castas mais tolerantes ao Zn e Cu. Os esfregaços cromossómicos mitóticos preparados a partir de raízes e folhas foram utilizados em coloração com nitrado de prata e hibridação in situ fluorescente (FISH) sequencial usando a sonda pTa71 (45S rDNA). Detetou-se um máximo de quatro nucléolos por interfase e de quatro loci rDNA por metafase, corroborando estudos anteriores. A técnica de coloração com nitrato de prata foi também aplicada no material vegetal colhido nos ensaios de hidroponia para verificar como as diferentes concentrações de Zn e Cu afetavam a atividade nucleolar nas diferentes castas. Irregularidades em células interfásicas bem como alterações no número e na área nucleolar foram observadas. Após a otimização das técnicas ensaio do cometa em condições alcalinas (“alkaline comet assay”) seguido de FISH (Comet-FISH), aplicadas pela primeira vez em V. vinifera, analisou-se o dano no DNA em núcleos de folhas desenvolvidas em estacas mantidas em ensaio hidropónico com soluções de Cu durante sete semanas. O ensaio do cometa revelou a presença de cometas de categorias um a quatro. A análise da distribuição dos sinais de hibridação das sondas pTa71 e telomérica ao longo da estrutura do cometa através da técnica Comet-FISH permitiu detetar dano parcial no DNA ribossomal (45S) e telomérico. Alguns sinais de toxicidade observados nas estacas de videira tratadas separadamente com concentrações crescentes de Zn e Cu foram semelhantes, nomeadamente, o aumento da espessura das raízes e a supressão do seu crescimento; a diminuição do índice mitótico e da atividade nucleolar nas raízes. As concentrações de Zn mais altas induziram ainda crescimento foliar acompanhado pela diminuição da espessura da lâmina foliar e uma redução na atividade nucleolar da folha. As raízes tratadas com Cu apresentaram um aumento da percentagem de células interfásicas e mitóticas com anomalias. Entre as castas analisadas após hidroponia realizada em soluções enriquecidas com Zn, considerou-se Donzelinho Branco e Verdelho como as mais tolerantes à toxicidade induzida pelo Zn. No grupo de castas analisadas nos ensaios hidropónicos com soluções de Cu, as castas Malvasia Fina e Viosinho revelaram-se mais tolerantes. Esta tese incluiu estudos de cito-, geno- e fitotoxicidade em resposta ao excesso de Zn e Cu que foram baseados em múltiplas técnicas aplicadas a diferentes castas cultivadas na Região Demarcada do Douro. Porém, as abordagens aqui utilizadas podem ser extensíveis a outras regiões vitivinícolas e castas, e podem contribuir para a seleção de castas mais tolerantes ao stress abiótico gerado pela toxicidade induzida pelo excesso de Zn, Cu ou outros micronutrientes/ metais pesados, no âmbito de ensaios de campo e/ou programas de melhoramento.
Agricultural soils composition has several heavy metals whose concentration differs between regions due to different physical-chemical properties. However, the application of agrochemicals based on heavy metals such as Zinc (Zn) and Copper (Cu) has led to the contamination of agricultural soils, in particular vineyards. These heavy metals are essential micronutrients for plant metabolic activities, but in reduced concentrations. The contamination of vineyard soils with Zn and Cu is even more exacerbated in soils with an acid pH (predominant in North of Portugal), since it increases Zn bioavailability in the soil and the translocation of Cu to plants, inducing cyto- , geno- and phytotoxicity. Due to the economic importance of the wine sector in Portugal, and the relevance and international recognition of wines produced in the Douro region, this thesis focused on the cytogenomic and morpho-histological analysis of roots and/or leaves of red and white grape varieties grown in this region. Given the difficulty in accessing grapevine roots, the harvested cuttings were forced and maintained in hydroponic experiments. Cuttings of the varieties ‘Donzelinho Branco’, ‘Verdelho’ and ‘Bastardo’ were used in a hydroponic experiment with nutrient solution enriched with different concentrations of Zn (1.5, 3 and 5 mM). Cuttings of the ‘Tinta Barroca’, ‘Tinto Cão’, ‘Malvasia Fina’ and ‘Viosinho’ varieties were used in a second hydroponic experiment with nutritive solution enriched with different Cu concentrations (1, 10, 25 and 50 µM). These concentrations of Zn and Cu were based on values previously determined for Douro soils or established by the European Union. Newly formed leaves and roots during hydroponics were used to optimize cytogenomic techniques that were applied for the first time in Vitis vinifera L. and that proved useful in the analysis of tolerance to different abiotic stresses in other plant species. The aim of this study was to integrate cytogenomic data with those obtained through morpho histological analyses in roots and/or leaves to determine which varieties are more tolerant to Zn and Cu. Mitotic chromosomal spreads prepared from roots and leaves were used in silver nitrate staining and sequential fluorescent in situ hybridization (FISH) with the probe pTa71 (45S rDNA). A maximum of four nucleoli per interphase and four rDNA loci per metaphase were detected, corroborating previous studies. The technique of silver nitrate staining was also applied to plant material collected in hydroponics tests to verify how the different concentrations of Zn and Cu affected the nucleolar activity in different varieties. Irregularities in interphasic cells, as well as alterations in the number and in the nucleolar area, were observed. After optimizing the techniques of alkaline comet assay followed by FISH (CometFISH), applied for the first time in V. vinifera, the DNA damage was analysed in nuclei of leaves developed in cuttings that were kept in hydroponic solutions enriched with Cu for seven weeks. The comet assay revealed the presence of comets from categories one to four. The analysis of the distribution of the pTa71 and telomeric probe hybridization signals along the comet structure with the comet-FISH technique allowed the detection of partial damage on the ribosomal (45S) and telomeric DNA. Some evidence of toxicity observed in the grapevine cuttings treated separately with increasing concentrations of Zn and Cu were similar, namely, the increase of root thickness and the suppression of its growth; and the decrease of the mitotic index and nucleolar activity in the roots. Higher Zn concentrations also induced leaf growth accompanied by a decrease in leaf blade thickness and a reduction in leaf nucleolar activity. The roots treated with Cu showed an increase in the percentage of interphase and mitotic cells with anomalies. Among the varieties analysed after hydroponics performed with solutions enriched with Zn, ‘Donzelinho Branco’ and ‘Verdelho’ were considered the most tolerant to Zn-induced toxicity. In the group of varieties analysed in the hydroponic experiment with Cu solutions, the ‘Malvasia Fina’ and ‘Viosinho’ varieties were more tolerant. This thesis included studies of cyto-, geno- and phytotoxicity in response to the excess of Zn and Cu that were based on multiple techniques applied to different grapevine varieties, cultivated in the Douro Demarcated Region. However, the approaches used here can be extended to other wine-producing regions and varieties and can contribute to the selection of varieties more tolerant to the abiotic stress generated by toxicity induced by the excess of Zn, Cu or other micronutrients/ heavy metals, within the scope of field trials and/ or breeding programs.
Agricultural soils composition has several heavy metals whose concentration differs between regions due to different physical-chemical properties. However, the application of agrochemicals based on heavy metals such as Zinc (Zn) and Copper (Cu) has led to the contamination of agricultural soils, in particular vineyards. These heavy metals are essential micronutrients for plant metabolic activities, but in reduced concentrations. The contamination of vineyard soils with Zn and Cu is even more exacerbated in soils with an acid pH (predominant in North of Portugal), since it increases Zn bioavailability in the soil and the translocation of Cu to plants, inducing cyto- , geno- and phytotoxicity. Due to the economic importance of the wine sector in Portugal, and the relevance and international recognition of wines produced in the Douro region, this thesis focused on the cytogenomic and morpho-histological analysis of roots and/or leaves of red and white grape varieties grown in this region. Given the difficulty in accessing grapevine roots, the harvested cuttings were forced and maintained in hydroponic experiments. Cuttings of the varieties ‘Donzelinho Branco’, ‘Verdelho’ and ‘Bastardo’ were used in a hydroponic experiment with nutrient solution enriched with different concentrations of Zn (1.5, 3 and 5 mM). Cuttings of the ‘Tinta Barroca’, ‘Tinto Cão’, ‘Malvasia Fina’ and ‘Viosinho’ varieties were used in a second hydroponic experiment with nutritive solution enriched with different Cu concentrations (1, 10, 25 and 50 µM). These concentrations of Zn and Cu were based on values previously determined for Douro soils or established by the European Union. Newly formed leaves and roots during hydroponics were used to optimize cytogenomic techniques that were applied for the first time in Vitis vinifera L. and that proved useful in the analysis of tolerance to different abiotic stresses in other plant species. The aim of this study was to integrate cytogenomic data with those obtained through morpho histological analyses in roots and/or leaves to determine which varieties are more tolerant to Zn and Cu. Mitotic chromosomal spreads prepared from roots and leaves were used in silver nitrate staining and sequential fluorescent in situ hybridization (FISH) with the probe pTa71 (45S rDNA). A maximum of four nucleoli per interphase and four rDNA loci per metaphase were detected, corroborating previous studies. The technique of silver nitrate staining was also applied to plant material collected in hydroponics tests to verify how the different concentrations of Zn and Cu affected the nucleolar activity in different varieties. Irregularities in interphasic cells, as well as alterations in the number and in the nucleolar area, were observed. After optimizing the techniques of alkaline comet assay followed by FISH (CometFISH), applied for the first time in V. vinifera, the DNA damage was analysed in nuclei of leaves developed in cuttings that were kept in hydroponic solutions enriched with Cu for seven weeks. The comet assay revealed the presence of comets from categories one to four. The analysis of the distribution of the pTa71 and telomeric probe hybridization signals along the comet structure with the comet-FISH technique allowed the detection of partial damage on the ribosomal (45S) and telomeric DNA. Some evidence of toxicity observed in the grapevine cuttings treated separately with increasing concentrations of Zn and Cu were similar, namely, the increase of root thickness and the suppression of its growth; and the decrease of the mitotic index and nucleolar activity in the roots. Higher Zn concentrations also induced leaf growth accompanied by a decrease in leaf blade thickness and a reduction in leaf nucleolar activity. The roots treated with Cu showed an increase in the percentage of interphase and mitotic cells with anomalies. Among the varieties analysed after hydroponics performed with solutions enriched with Zn, ‘Donzelinho Branco’ and ‘Verdelho’ were considered the most tolerant to Zn-induced toxicity. In the group of varieties analysed in the hydroponic experiment with Cu solutions, the ‘Malvasia Fina’ and ‘Viosinho’ varieties were more tolerant. This thesis included studies of cyto-, geno- and phytotoxicity in response to the excess of Zn and Cu that were based on multiple techniques applied to different grapevine varieties, cultivated in the Douro Demarcated Region. However, the approaches used here can be extended to other wine-producing regions and varieties and can contribute to the selection of varieties more tolerant to the abiotic stress generated by toxicity induced by the excess of Zn, Cu or other micronutrients/ heavy metals, within the scope of field trials and/ or breeding programs.
Descrição
Tese de Doutoramento em Ciências Químicas e Biológicas
Palavras-chave
Citogenética , citotoxicidade