Ecofisiologia da cerejeira (Prunus avium L.),composição fenólica e actividade antioxidante dos frutos
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2006
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No contexto da fruticultura moderna, a cerejeira (Prunus avium L.) tem uma elevada importância económica, havendo, por isso, a preocupação especial de formar árvores produtoras de cerejas com elevada qualidade, mas com porte mais ananicante devido aos elevados custos da mão-de-obra na colheita e noutras práticas culturais do pomar. Assim, a expansão e a exploração rentável desta cultura implicam a aquisição de conhecimentos, entre outros, nos domínios da ecofisiologia das novas combinações cultivar/porta-enxerto e das características físico-químicas dos frutos. Para isso, traçaram-se três objectivos principais: (1) Estudar a ecofisiologia da cerejeira em 15 combinações cultivar/porta-enxerto, ao nível das características morfo-anatómicas, bioquímicas e fisiológicas de árvores ananicantes versus árvores vigorosas; (2) Estudar o efeito da cultivar, do estado de maturação, do ano e da conservação pós-colheita na qualidade sensorial e nutricional da cereja, através de análises físico-químicas (principalmente de compostos fenólicos); (3) Avaliar o potencial antioxidante dos extractos de cereja, à colheita e depois da conservação.
Os estudos no âmbito deste trabalho incidiram sobre quatro cultivares de cerejeira, a Burlat, a Saco, a Summit e a Van, por produzirem frutos com boa aptidão para consumo em fresco e terem elevado potencial produtivo. As cultivares foram enxertadas em cinco porta¬enxertos, com efeito ananicante crescente: Prunus avium < CAB 11E < Maxma 14 < Gisela 5 < Edabriz. As características gerais dos porta-enxertos e das cultivares encontram-se nos Anexo I e II, respectivamente. A parte experimental deste estudo decorreu basicamente durante 2001–2003, num pomar de cerejeiras instalado em 1999, sito em Abambres, Vila Real. Refere-se, no entanto, que se fez o acompanhamento do ensaio desde a instalação dos porta-enxertos no viveiro em 1997, da sua enxertia em 1998 e posterior plantação em 1999. Os resultados obtidos estão estruturados em oito capítulos, sete dos quais (capítulos 2–8), incluem integralmente a informação publicada ou submetida em revistas indexadas na lista do Journal Citation Reports.
O capítulo 2 descreve o estudo cujo principal objectivo foi avaliar os efeitos do atarraque do porta-enxerto e da densidade de plantação no crescimento e nas relações hídricas das cerejeiras. Em 1997, iniciou-se um ensaio com os cinco porta-enxertos sujeitos a diferentes intensidades de atarraque (0, 30, 60 e 90% do crescimento vegetativo foi removido, designando-se por P1, P2, P3 e P4, respectivamente) após terem sido plantados em dois compassos de plantação (S1 = 0,25 x 1,0 m e S2 = 0,45 x 1,5 m). O crescimento vegetativo e o potencial hídrico foliar foram quantificados durante a estação de crescimento. Dos resultados obtidos, concluiu-se que o atarraque à plantação afectou o crescimento do sistema radicular dos porta-enxertos. O não atarraque (P1) originou plantas com maior comprimento e peso do sistema radicular face às plantas com atarraque mais intenso (P4). Por sua vez, a combinação S1/P1 permitiu a obtenção de uma maior biomassa radicular, enquanto S1/P4 e S2/P4 deram origem às menores biomassas radiculares. O potencial hídrico foliar (Ψfolha) variou significativamente entre os porta-enxertos e com o compasso de plantação, mas não com o atarraque. Os porta-enxertos Maxma 14 e Prunus avium atingiram os valores mais negativos de Ψfolha ao meio-dia, mas os maiores valores antes do amanhecer. Em geral, os porta-enxertos apresentaram Ψfolha mais negativos no compasso mais apertado.
No capítulo 3 pretendeu-se estudar a arquitectura hidráulica e as limitações ao transporte da água em combinações cultivar/porta-enxerto. Para tal escolheu-se a cultivar Van enxertada nos cinco porta-enxertos e avaliou-se a anatomia do xilema, a condutividade hidráulica relativa (RC) e o índice de vulnerabilidade (VI) de raízes de diâmetro < 2 mm, 2–5 mm e > 5 mm e de caules. Foram ainda determinadas as relações hídricas, trocas gasosas e variações no crescimento. As raízes apresentaram vasos xilémicos de maior diâmetro (VD), maior RC e VI do que os caules das 5 combinações Van/porta-enxertos. Também se verificou uma maior frequência de vasos xilémicos (VF), mas menor VD, RC e VI das árvores ananicantes, i.e., enxertadas em Edabriz e Gisela 5, do que as árvores nos porta-enxertos mais vigorosos Prunus avium, CAB 11E e Maxma 14. Imposições anatómicas provocadas pelos vasos xilémicos de menor diâmetro no estado de hidratação das árvores ananicantes conduzem a uma série de feedbacks negativos, tais como uma redução na RC, Ψ, trocas gasosas e crescimento.
No capítulo 4 procurou-se estudar o efeito da arquitectura da copa nas variações morfo-anatómicas (massa foliar por unidade de área – LMA, densidade da folha – LD, espessura dos diferentes tecidos e densidade estomática), composição química (pigmentos fotossintéticos, açúcares solúveis totais – SS, amido – St e fenóis totais – TP), trocas gasosas e potencial hídrico do caule (Ψcaule) verificadas em folhas das cultivares Burlat e Summit (porte erecto, copas muito densas) e Van (porte semi-erecto, copa menos densa). De facto, este estudo indicou que a copa da Van favoreceu uma maior transmitância de luz, com efeitos positivos na taxa fotossintética, especialmente à colheita. No entanto, não se registaram diferenças no estado hídrico das três cultivares. As folhas da Van tinham maior LMA e as variações neste parâmetro estiveram sobretudo associadas à maior espessura do mesófilo e não à respectiva densidade. Anatomicamente, as folhas da Summit e da Van eram mais espessas do que as da Burlat, principalmente devido à maior espessura do mesófilo, embora com menor razão parênquima em paliçada/lacunoso do que a Burlat. A Van tinha a maior concentração de clorofila total (Chl) por unidade área e a maior razão Chla/b. Por outro lado, a Van apresentou as menores concentrações de SS, St e TP.
No capítulo 5, cujo objectivo era relacionar diversos parâmetros fisiológicos das folhas (relações hídricas, trocas gasosas, fluorescência da clorofila a, transmitância da luz pela copa, pigmentos fotossintéticos e metabolitos) e da qualidade dos frutos (peso, cor, firmeza, pH, índice refractométrico e acidez titulável) em cultivares (Burlat, Summit e Van) enxertadas nos cinco porta-enxertos, constatou-se que o porta-enxerto afectou significativamente todos os parâmetros fisiológicos. Com efeito, as cerejeiras enxertadas em porta-enxertos mais vigorosos apresentaram maior potencial hídrico ao meio-dia (ΨMD), taxa de assimilação líquida de CO2 (A), condutância estomática (gs), concentração de CO2 intercelular (Ci) e eficiência fotoquímica máxima do fotossistema II (Fv/Fm). O ΨMD correlacionou-se positivamente com A, gs e Ci. Também A foi correlacionada positivamente com gs e os declives das regressões lineares e os respectivos coeficientes de determinação aumentaram dos porta-enxertos mais vigorosos para os mais ananicantes. O efeito do porta¬enxerto foi também significativo para os pigmentos fotossintéticos, enquanto que as concentrações de metabolitos e as características físico-químicas dos frutos mostraram-se mais dependentes do efeito da cultivar. Das três cultivares em estudo, as folhas da Burlat tinham as menores concentrações de pigmentos fotossintéticos, mas eram as mais ricas em SS, St e TP. Comparativamente às outras cultivares, a Summit apresentou frutos mais pesados e maiores, independentemente do porta-enxerto. A Burlat revelou frutos menos firmes e com menor concentração de SS e acidez titulável do que os da Van. No entanto, as cerejas da Van tinham menor luminosidade (L*), croma (C*) e tonalidade (H*), o que é indicativo de frutos mais vermelhos e escuros, comparativamente aos da Summit. De uma maneira geral, o ΨMD foi correlacionado positivamente com o peso do fruto e A foi correlacionada negativamente com a L* e C* do fruto.
O capítulo 6 centrou-se no estudo do efeito da cultivar, do estado de maturação (cerejas parcialmente maduras e no estado óptimo de maturação), da conservação (temperatura ambiente – T1, 15 ± 5 ºC e 55–60% H.R., durante uma semana; câmara frigorífica, em atmosfera normal – T2, 1–2 ºC e 90–95% H.R., durante um mês) e do ano (condições climáticas, nomeadamente temperatura e insolação) no perfil fenólico dos frutos das quatro cultivares de cerejeira. Dos resultados obtidos, constatou-se que os ácidos hidroxicinâmicos mais abundantes em todas as cultivares foram o ácido neoclorogénico e o ácido p-cumaroilquínico e, em concentração menos elevada, o ácido clorogénico. A cianidina¬3-rutinósido e a cianidina-3-glucósido foram identificadas como sendo as principais antocianinas das cerejas. Em contrapartida, a peonidina e a pelargonidina-3-rutinósido tiveram pouca expressão. A peonidina-3-glucósido apenas foi identificada nas cultivares Burlat e Van. Foram ainda identificados dois flavanóis, em que os compostos deste grupo fenólico mais quantificados foram a epicatequina e a catequina. O flavonol rutina foi também detectado. A cultivar Saco registou as concentrações mais elevadas de fenóis totais (227 mg 100 g–1 de peso fresco), contrastando com a Van (124 mg 100 g–1 de peso fresco). As concentrações de compostos fenólicos foram sempre mais altas em cerejas maduras do que em cerejas parcialmente maduras. A conservação provocou variações nos ácidos hidroxicinâmicos, sendo a tendência final uma redução das concentrações nas cerejas armazenadas a T2 e um aumento nas cerejas armazenadas para todas as cultivares a T1, excepto para a Burlat. Os teores em antocianinas aumentaram em ambos os dois estados de conservação, especialmente na Van, em que o aumento quintuplicou no armazenamento a T1 (de 47 para 230 mg 100 g–1 de peso fresco). As concentrações de rutina, epicatequina e catequina permaneceram bastante estáveis durante o armazenamento em ambas as temperaturas. Geralmente, registaram-se maiores concentrações de ácidos hidroxicinâmicos em 2001 e de antocianinas em 2002.
No capítulo 7 estudou-se a relação entre os parâmetros cromáticos e a concentração de antocianinas nos frutos das quatro cultivares de cerejeira analisadas à colheita e após conservação em T1 e T2. A cor foi determinada através de medição colorimétrica (sistema CIELAB) directamente nos frutos, enquanto que as antocianinas foram quantificadas por HPLC-DAD. A concentração de antocianinas totais dos frutos das diferentes cultivares variou na seguinte ordem: Burlat > Saco > Van > Summit. As cerejas armazenadas em T1 apresentam maior redução da L*, C* e H* do que os frutos armazenados em T2. Os parâmetros cromáticos (L*, a*, b*, C* e H*) correlacionaram-se negativamente com o teor em antocianinas totais e com os fenóis totais. Os resultados permitem concluir que as funções cromáticas do C* e H* podem servir para prever a evolução da cor e os níveis de antocianinas durante o período de conservação.
No capítulo 8 procurou-se avaliar a actividade antioxidante dos extractos de cerejas das quatro cultivares, à colheita e depois da conservação em T1 e T2. Os ácidos hidroxicinâmicos dominaram em todos os extractos e representaram 60–74% dos compostos fenólicos das cerejas frescas ou armazenadas das cultivares Saco, Summit e Van, e apenas 45% dos compostos fenólicos da Burlat. Os compostos fenólicos dos extractos das cerejas inibiram a oxidação das lipoproteínas de baixa densidade (LDL) in vitro. À medida que se aumentava a concentração de compostos fenólicos aumentava a actividade antioxidante. A adição de 20 µM de equivalentes de ácido gálico (GAE) bloqueou integralmente a oxidação das LDL in vitro. Quando comparados a 12,5 µM GAE, os extractos das cerejas frescas tiveram maior actividade antioxidante. Os frutos das cultivares Summit e Van tiveram a maior e a menor actividade antioxidante, respectivamente. As cerejas Van conservadas em T1 e T2 manifestaram actividade prooxidante. As diferenças no efeito antioxidante foram positivamente correlacionadas com as concentrações do ácido p-cumaroilquínico, mas negativamente correlacionadas com a cianidina-3-rutinósido.
Finalmente, no capítulo 9 apresentam-se as conclusões mais relevantes e sugerem-se temas para trabalho futuro.
In modern horticulture, sweet cherry tree (Prunus avium L.) is economically very important. The actual trends of sweet cherry tree culture are to obtain dwarf trees and high fruit quality for fresh market. Therefore, the cherry tree management involves the knowledge about the ecophysiology of the new scion-rootstock combinations and the physicochemical fruit characteristics. So, three main objectives were considered: (1) To study the ecophysiology of fifteen scion-rootstock combinations at morpho-anatomical, biochemical and physiological level of dwarfed trees versus invigorating trees; (2) To study the effect of cultivar, ripeness stage, postharvest storage and year in the nutritional fruit quality, by physicochemical analysis (mainly of phenolic compounds); (3) To evaluate the antioxidant activity of cherry extracts at harvest and after storage. The study was carried out with four high quality sweet cherry cultivars Burlat, Saco, Summit and Van grafted onto five rootstocks with contrasting size-controlling potentials, which increase in the order: Prunus avium < CAB 11E < Maxma 14 < Gisela 5 < Edabriz. The experimental study was conducted essentially during 2001–2003 in a sweet cherry tree orchard installed in 1999, in Abambres, Vila Real. However, a preliminary study was done with the rootstocks in the nursery in 1997, during grafting in 1998 and after plantation in 1999. The experimental work is divided in eight chapters, seven of them (chapters 2–8) include the information published or submitted to journals belonging to the Journal Citation Reports. In chapter 2, the aim of the first study was to describe the effects of pruning and plant spacing on growth and water relations of sweet cherry trees. In the nursery, a trial with the five cherry rootstocks was begun in 1997. Pruning severities were applied on the rootstocks (0, 30, 60 and 90% of the vegetative growth was removed corresponding to P1, P2, P3 and P4 treatments, respectively) after planting to two plant spacings (S1 = 0.25 x 1.0 m and S2 = 0.45 x 1.5 m). Canopy, root growth and leaf water potential (Ψleaf) were quantified throughout the growing season. Pruning significantly affected root length and root weight of the rootstocks. Uncut plants (P1) showed a heavier and expanded root biomass than the intensively pruned plants (P4). The greater root biomass was obtained with the spacing/pruning combination, S1/P1, and the smaller with S1/P4 and S2/P4. Ψleaf varied significantly between the rootstocks and plant spacing but not with pruning. Maxma 14 and Prunus avium attained the lowest values of midday Ψleaf, but the highest values of predawn Ψleaf. Generally, with high density (S1), the rootstocks exhibited lower predawn and midday Ψleaf. In chapter 3, to assess hydraulic architecture and limitations to water transport across scion-rootstock combinations (Prunus avium L. cultivar Van grafted on the five rootstocks), we compared xylem anatomy, relative hydraulic conductivity (RC) and vulnerability index (VI) of roots (small diameter, < 2 mm; medium, 2–5 mm; and large, > 5 mm) and stems. Water relations, leaf gas exchange and variations in growth were also determined. Roots exhibited larger-diameter xylem conduits (VD), greater RC and VI than stems in all Van-rootstock combinations. Moreover, the data obtained here showed significantly higher vessel frequency (VF), lower VD, RC and VI in dwarfed trees, i.e., grafted on Edabriz and Gisela 5 than trees on the invigorating rootstocks Prunus avium, CAB 11E and Maxma 14. Anatomical constraints on turgor imposed by the smaller VD of dwarfed trees imply a series of negative feedbacks, like a decrease in RC, stem water potential (Ψstem), leaf gas exchange and growth. In chapter 4, canopy architecture effects on morpho-anatomy (leaf mass per unit area – LMA, leaf density – LD, leaf tissue thickness and stomatal density), chemical composition (photosynthetic pigments, total soluble sugars – SS, starch – St and total phenols – TP concentrations), gas exchange rates and Ψstem were studied in leaves of Prunus avium L. cultivars Burlat, Summit (upright, high dense canopies) and Van (spreading, low dense canopy). Van presented the highest value of canopy light transmittance, which improves photosynthetic rates, especially at harvest, compared to Summit and Burlat. However, no differences in Ψstem were found among cultivars. Van showed the highest LMA and variations in this parameter were mainly associated with alterations in leaf thickness than in LD. Anatomically, leaves of Summit and Van were thicker than those of Burlat, mainly due to increased mesophyll thickness, but they had lower palisade/spongy ratio. Total chlorophyll (Chl) concentration per area and the Chla/b ratio were always higher in Van leaves than in the other two cultivars. On the other hand, Van leaves had the lowest concentrations of SS, St and TP. In chapter 5, water relations, leaf gas exchange, chlorophyll a fluorescence, canopy light transmittance, leaf photosynthetic pigments and metabolites and fruit quality indices (weight, colour, firmness, pH, soluble solids and titratable acidity) of cherries from cultivars Burlat, Summit and Van growing on the five rootstocks were studied. Rootstock genotype affected all physiological parameters. Cherry cultivars grafted on invigorating rootstocks had higher values of midday stem water potential (ΨMD), net CO2 assimilation rate (A), stomatal conductance (gs), intercellular CO2 concentration (Ci) and maximum photochemical efficiency of photosystem II (PSII) (Fv/Fm) than cultivars grafted on dwarfing rootstocks. The ΨMD was positively correlated with A, gs and Ci. Moreover, A was positively correlated with gs, and the slopes of the linear regression and the respective coefficients of determination increased from invigorating to dwarfing rootstocks. The effect of rootstock genotype was also statistically significant for leaf photosynthetic pigments, whereas metabolite concentrations and fruit physicochemical characteristics were more dependent on cultivar genotype. Among cultivars, Burlat leaves had the lowest concentrations of photosynthetic pigments, but were richest in SS, St and TP. Compared with the other cultivars, Summit had heavier fruits, independent of the rootstock. Burlat cherries were less firm and had lower concentrations of soluble sugars and lower titratable acidity than Van cherries. Nevertheless, Van cherries had lower lightness (L*), chroma (C*) and hue angle (H*), representing redder and darker cherries, compared with Summit fruits. In general, ΨMD was positively correlated with fruit mass and A was negatively correlated with L* and C*. In chapter 6 the main objective was to study the effect of cultivar, ripeness stage (partially ripe and ripe), postharvest storage (room temperature – T1, 15 ± 5 °C and 55−60% R.H., for one week; and cool temperature – T2, 1–2 °C and 90−95% R.H., for one month) and year (climatic conditions, namely, temperature and sunshine hours) in the phenolic profile of the cherries from the four sweet cherry cultivars. Neochlorogenic and p¬coumaroylquinic acids were the main hydroxycinnamic acid derivatives, but chlorogenic acid was also identified in all cultivars. The 3-glucoside and 3-rutinoside of cyanidin were the major anthocyanins. Peonidin and pelargonidin 3-rutinosides were the minor anthocyanins, and peonidin-3-glucoside was also present in Burlat and Van. Epicatechin was the main monomeric flavan-3-ol with catechin present in smaller amounts in all cultivars. The flavonol rutin was also detected. Saco contained the highest amounts of phenolics [227 mg 100 g–1 of fresh weight (fw)] and Van the lowest (124 mg 100 g–1 of fw). Levels of phenolics were always higher in ripe than in partially ripe cherries. Phenolic acid contents generally decreased with storage at T2 and increased with storage at T1. Anthocyanin levels increased at both storage temperatures. In Van the anthocyanins increased up to 5-fold during storage at 15 ± 5 °C (from 47 to 230 mg 100 g–1 of fw). Rutin, epicatechin and catechin contents remained quite constant at both storage conditions. In general, for all cultivars the levels of phenolic acids were higher in 2001 and the anthocyanin levels were higher in 2002. In chapter 7, the relationship between colour parameters and anthocyanins of the four sweet cherry cultivars was studied at harvest, and after storage at T1 and T2. The colour was measured by tristimulus colourimetry (CIELAB system) directly on the fruits, while anthocyanins were quantified by HPLC-DAD analysis on methanolic extracts of freeze-dried samples of the fresh cherries and on the differently stored cherries. The total anthocyanin content in fruits of the different cultivars varied in the order Burlat > Saco > Van > Summit. Cherries stored at T1 showed higher reduction of L*, C* and H* than fruits stored at T2. Chromatic parameters (L*, a*, b*, C* and H*) correlated negatively with the total anthocyanins levels and with the total phenols. The results show that chromatic functions of C* and H* can be used to predict the evolution of colour and anthocyanins levels during storage of sweet cherries. In chapter 8, the antioxidant activity of the phenolic extracts of the four sweet cherry cultivars were analysed at harvest and after storage at T1 and T2. Hydroxycinnamates dominated in all samples and represented 60–74% by weight of the phenols in the fresh and stored samples of the cultivars Saco, Summit and Van, and 45% by weight of the phenols in the cultivar Burlat samples, which were richer in anthocyanins. Phenolic cherry extracts inhibited low-density lipoprotein (LDL) oxidation in vitro in a dose-dependent manner. Addition of 20 µM gallic acid equivalents (GAE) completely blocked the oxidation in the LDL in vitro. When compared at equimolar addition levels of 12.5 µM GAE the extracts of freshly harvested cherries exhibited significantly higher antioxidant activities than extracts of stored samples. Summit samples had the highest antioxidant activity. Van storage at both T1 and T2 led to a prooxidant activity of the extracts. Differences in the antioxidant effects of the cherry samples were positively correlated with their levels of p-coumaroylquinic acid but negatively correlated with their cyanidin-3-rutinoside levels. Finally, in chapter 9 is presented the main conclusions of the study and proposals for future research.
In modern horticulture, sweet cherry tree (Prunus avium L.) is economically very important. The actual trends of sweet cherry tree culture are to obtain dwarf trees and high fruit quality for fresh market. Therefore, the cherry tree management involves the knowledge about the ecophysiology of the new scion-rootstock combinations and the physicochemical fruit characteristics. So, three main objectives were considered: (1) To study the ecophysiology of fifteen scion-rootstock combinations at morpho-anatomical, biochemical and physiological level of dwarfed trees versus invigorating trees; (2) To study the effect of cultivar, ripeness stage, postharvest storage and year in the nutritional fruit quality, by physicochemical analysis (mainly of phenolic compounds); (3) To evaluate the antioxidant activity of cherry extracts at harvest and after storage. The study was carried out with four high quality sweet cherry cultivars Burlat, Saco, Summit and Van grafted onto five rootstocks with contrasting size-controlling potentials, which increase in the order: Prunus avium < CAB 11E < Maxma 14 < Gisela 5 < Edabriz. The experimental study was conducted essentially during 2001–2003 in a sweet cherry tree orchard installed in 1999, in Abambres, Vila Real. However, a preliminary study was done with the rootstocks in the nursery in 1997, during grafting in 1998 and after plantation in 1999. The experimental work is divided in eight chapters, seven of them (chapters 2–8) include the information published or submitted to journals belonging to the Journal Citation Reports. In chapter 2, the aim of the first study was to describe the effects of pruning and plant spacing on growth and water relations of sweet cherry trees. In the nursery, a trial with the five cherry rootstocks was begun in 1997. Pruning severities were applied on the rootstocks (0, 30, 60 and 90% of the vegetative growth was removed corresponding to P1, P2, P3 and P4 treatments, respectively) after planting to two plant spacings (S1 = 0.25 x 1.0 m and S2 = 0.45 x 1.5 m). Canopy, root growth and leaf water potential (Ψleaf) were quantified throughout the growing season. Pruning significantly affected root length and root weight of the rootstocks. Uncut plants (P1) showed a heavier and expanded root biomass than the intensively pruned plants (P4). The greater root biomass was obtained with the spacing/pruning combination, S1/P1, and the smaller with S1/P4 and S2/P4. Ψleaf varied significantly between the rootstocks and plant spacing but not with pruning. Maxma 14 and Prunus avium attained the lowest values of midday Ψleaf, but the highest values of predawn Ψleaf. Generally, with high density (S1), the rootstocks exhibited lower predawn and midday Ψleaf. In chapter 3, to assess hydraulic architecture and limitations to water transport across scion-rootstock combinations (Prunus avium L. cultivar Van grafted on the five rootstocks), we compared xylem anatomy, relative hydraulic conductivity (RC) and vulnerability index (VI) of roots (small diameter, < 2 mm; medium, 2–5 mm; and large, > 5 mm) and stems. Water relations, leaf gas exchange and variations in growth were also determined. Roots exhibited larger-diameter xylem conduits (VD), greater RC and VI than stems in all Van-rootstock combinations. Moreover, the data obtained here showed significantly higher vessel frequency (VF), lower VD, RC and VI in dwarfed trees, i.e., grafted on Edabriz and Gisela 5 than trees on the invigorating rootstocks Prunus avium, CAB 11E and Maxma 14. Anatomical constraints on turgor imposed by the smaller VD of dwarfed trees imply a series of negative feedbacks, like a decrease in RC, stem water potential (Ψstem), leaf gas exchange and growth. In chapter 4, canopy architecture effects on morpho-anatomy (leaf mass per unit area – LMA, leaf density – LD, leaf tissue thickness and stomatal density), chemical composition (photosynthetic pigments, total soluble sugars – SS, starch – St and total phenols – TP concentrations), gas exchange rates and Ψstem were studied in leaves of Prunus avium L. cultivars Burlat, Summit (upright, high dense canopies) and Van (spreading, low dense canopy). Van presented the highest value of canopy light transmittance, which improves photosynthetic rates, especially at harvest, compared to Summit and Burlat. However, no differences in Ψstem were found among cultivars. Van showed the highest LMA and variations in this parameter were mainly associated with alterations in leaf thickness than in LD. Anatomically, leaves of Summit and Van were thicker than those of Burlat, mainly due to increased mesophyll thickness, but they had lower palisade/spongy ratio. Total chlorophyll (Chl) concentration per area and the Chla/b ratio were always higher in Van leaves than in the other two cultivars. On the other hand, Van leaves had the lowest concentrations of SS, St and TP. In chapter 5, water relations, leaf gas exchange, chlorophyll a fluorescence, canopy light transmittance, leaf photosynthetic pigments and metabolites and fruit quality indices (weight, colour, firmness, pH, soluble solids and titratable acidity) of cherries from cultivars Burlat, Summit and Van growing on the five rootstocks were studied. Rootstock genotype affected all physiological parameters. Cherry cultivars grafted on invigorating rootstocks had higher values of midday stem water potential (ΨMD), net CO2 assimilation rate (A), stomatal conductance (gs), intercellular CO2 concentration (Ci) and maximum photochemical efficiency of photosystem II (PSII) (Fv/Fm) than cultivars grafted on dwarfing rootstocks. The ΨMD was positively correlated with A, gs and Ci. Moreover, A was positively correlated with gs, and the slopes of the linear regression and the respective coefficients of determination increased from invigorating to dwarfing rootstocks. The effect of rootstock genotype was also statistically significant for leaf photosynthetic pigments, whereas metabolite concentrations and fruit physicochemical characteristics were more dependent on cultivar genotype. Among cultivars, Burlat leaves had the lowest concentrations of photosynthetic pigments, but were richest in SS, St and TP. Compared with the other cultivars, Summit had heavier fruits, independent of the rootstock. Burlat cherries were less firm and had lower concentrations of soluble sugars and lower titratable acidity than Van cherries. Nevertheless, Van cherries had lower lightness (L*), chroma (C*) and hue angle (H*), representing redder and darker cherries, compared with Summit fruits. In general, ΨMD was positively correlated with fruit mass and A was negatively correlated with L* and C*. In chapter 6 the main objective was to study the effect of cultivar, ripeness stage (partially ripe and ripe), postharvest storage (room temperature – T1, 15 ± 5 °C and 55−60% R.H., for one week; and cool temperature – T2, 1–2 °C and 90−95% R.H., for one month) and year (climatic conditions, namely, temperature and sunshine hours) in the phenolic profile of the cherries from the four sweet cherry cultivars. Neochlorogenic and p¬coumaroylquinic acids were the main hydroxycinnamic acid derivatives, but chlorogenic acid was also identified in all cultivars. The 3-glucoside and 3-rutinoside of cyanidin were the major anthocyanins. Peonidin and pelargonidin 3-rutinosides were the minor anthocyanins, and peonidin-3-glucoside was also present in Burlat and Van. Epicatechin was the main monomeric flavan-3-ol with catechin present in smaller amounts in all cultivars. The flavonol rutin was also detected. Saco contained the highest amounts of phenolics [227 mg 100 g–1 of fresh weight (fw)] and Van the lowest (124 mg 100 g–1 of fw). Levels of phenolics were always higher in ripe than in partially ripe cherries. Phenolic acid contents generally decreased with storage at T2 and increased with storage at T1. Anthocyanin levels increased at both storage temperatures. In Van the anthocyanins increased up to 5-fold during storage at 15 ± 5 °C (from 47 to 230 mg 100 g–1 of fw). Rutin, epicatechin and catechin contents remained quite constant at both storage conditions. In general, for all cultivars the levels of phenolic acids were higher in 2001 and the anthocyanin levels were higher in 2002. In chapter 7, the relationship between colour parameters and anthocyanins of the four sweet cherry cultivars was studied at harvest, and after storage at T1 and T2. The colour was measured by tristimulus colourimetry (CIELAB system) directly on the fruits, while anthocyanins were quantified by HPLC-DAD analysis on methanolic extracts of freeze-dried samples of the fresh cherries and on the differently stored cherries. The total anthocyanin content in fruits of the different cultivars varied in the order Burlat > Saco > Van > Summit. Cherries stored at T1 showed higher reduction of L*, C* and H* than fruits stored at T2. Chromatic parameters (L*, a*, b*, C* and H*) correlated negatively with the total anthocyanins levels and with the total phenols. The results show that chromatic functions of C* and H* can be used to predict the evolution of colour and anthocyanins levels during storage of sweet cherries. In chapter 8, the antioxidant activity of the phenolic extracts of the four sweet cherry cultivars were analysed at harvest and after storage at T1 and T2. Hydroxycinnamates dominated in all samples and represented 60–74% by weight of the phenols in the fresh and stored samples of the cultivars Saco, Summit and Van, and 45% by weight of the phenols in the cultivar Burlat samples, which were richer in anthocyanins. Phenolic cherry extracts inhibited low-density lipoprotein (LDL) oxidation in vitro in a dose-dependent manner. Addition of 20 µM gallic acid equivalents (GAE) completely blocked the oxidation in the LDL in vitro. When compared at equimolar addition levels of 12.5 µM GAE the extracts of freshly harvested cherries exhibited significantly higher antioxidant activities than extracts of stored samples. Summit samples had the highest antioxidant activity. Van storage at both T1 and T2 led to a prooxidant activity of the extracts. Differences in the antioxidant effects of the cherry samples were positively correlated with their levels of p-coumaroylquinic acid but negatively correlated with their cyanidin-3-rutinoside levels. Finally, in chapter 9 is presented the main conclusions of the study and proposals for future research.
Descrição
Tese de Doutoramento em Engenharia Biológica e Ambiental
Palavras-chave
Prunus avium L. , Cerejeira , variedade , porta-enxerto , cereja , propriedade físico-química , Ecofisiologia , Compostos fenólicos