Postprandial plasma carotenoid responses following consumption of strawberries, red wine, vitamin C or spinach by elderly women

This study investigated the postprandial plasma responses of carotenoids for 24 h after feeding five specific breakfast beverages; four of which had low or no carotenoid content. In seven fasting healthy elderly female subjects a blood sample (baseline) was obtained, after which they were given a breakfast beverage, containing one of the following: 1) strawberries (240 g); 2) ascorbic acid (1250 mg); 3) spinach (294 g); 4) red wine (300 mL); and 5) control (breakfast beverage only). Blood samples were collected at 0.5, 1, 4, 7, 11, 15 and 24 h. Plasma carotenoids were measured using HPLC. No significant differences were found in the levels of the plasma carotenoids measured among the various treatments at baseline. In the spinach treatment, plasma lutein, zeaxanthin and β-carotene levels at 7, 11, 15 and 24 h were significantly higher than those at baseline, as expected. All of the carotenoids measured in the control and vitamin C treatments, at subsequent sampling times were not significantly different from those at baseline. However, for most carotenoids, strawberry and red wine feeding resulted in significantly lower carotenoids values from baseline at 11 and 15 h. Subjects who received a diet with low levels of carotenoids, but whose postprandial plasma levels of carotenoids remain steady, might be explained by a mechanism that promotes secretion of carotenoids into the circulation. Assuming that plasma carotenoids are being used over time, we hypothesize that strawberries and red wine contain some substances that interfere with the secretion of carotenoids into the circulation.

Qualidade e conservação do morango tratado em pós-colheita com cloreto de cálcio e do armazenamento em atmosfera modificada ativa

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of solvent effect on the extraction of phenolic compounds and antioxidant capacities from the berries: application of principal component analysis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Potencial de resíduos agroindustriais como fontes de compostos bioativos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Desempenho de cultivares de morango submetidas a diferentes tipos de polinização em cultivo protegido

Resumo – O objetivo deste trabalho foi avaliar o efeito de diferentes tipos de polinização sobre a qualidade de frutos de cultivares de morangueiro e sua contribuição isolada para a massa dos frutos, bem como determinar o potencial de Plebeia nigriceps (Hymenoptera: Apidae, Meliponini) como agente polinizador em ambiente protegido. As cultivares Aromas, Diamante e Cegnidarem foram submetidas a tratamentos com autopolinização, polinização por P. nigriceps e polinização livre. Os experimentos foram conduzidos em estufa tipo pampeana, coberta com polietileno transparente e desprovida de telas anti-insetos nas laterais, com 1.344 plantas. Para as avaliações, foram marcadas 56 flores primárias em botão, de cada cultivar, e considerou-se cada planta uma repetição. Avaliaram-se massa de matéria fresca, peso, diâmetro, comprimento e presença de deformação nos frutos. A polinização entomófila tem contribuição variada à massa dos frutos, de acordo com a cultivar. As cultivares apresentam sensibilidade variada à autopolinização, no que se refere à incidência de frutos deformados. Ainterferência da polinização entomófila na produtividade do morangueiro está mais relacionada à redução do percentual de frutos deformados do que ao aumento da massa dos frutos em si. O comportamento de P. nigriceps indica que a espécie apresenta potencial para polinização da cultura do morangueiro em ambiente protegido.

Desempenho de cultivares de morango submetidas a diferentes tipos de polinização em cultivo protegido

O objetivo deste trabalho foi avaliar o efeito de diferentes tipos de polinização sobre a qualidade de frutos de cultivares de morangueiro e sua contribuição isolada para a massa dos frutos, bem como determinar o potencial de Plebeia nigriceps (Hymenoptera: Apidae, Meliponini) como agente polinizador em ambiente protegido. As cultivares Aromas, Diamante e Cegnidarem foram submetidas a tratamentos com autopolinização, polinização por P. nigriceps e polinização livre. Os experimentos foram conduzidos em estufa tipo pampeana, coberta com polietileno transparente e desprovida de telas anti-insetos nas laterais, com 1.344 plantas. Para as avaliações, foram marcadas 56 flores primárias em botão, de cada cultivar, e considerou-se cada planta uma repetição. Avaliaram-se massa de matéria fresca, peso, diâmetro, comprimento e presença de deformação nos frutos. A polinização entomófila tem contribuição variada à massa dos frutos, de acordo com a cultivar. As cultivares apresentam sensibilidade variada à autopolinização, no que se refere à incidência de frutos deformados. A interferência da polinização entomófila na produtividade do morangueiro está mais relacionada à redução do percentual de frutos deformados do que ao aumento da massa dos frutos em si. O comportamento de P. nigriceps indica que a espécie apresenta potencial para polinização da cultura do morangueiro em ambiente protegido.

Armazenamento refrigerado de morango submetido a altas concentrações de CO2

Temperatura de 0ºC associada a atmosferas com 12 a 20% de CO2 têm sido recomendadas como condição ideal para o armazenamento de morango. Entretanto, as redes de distribuição e comercialização de produtos hortícolas no Brasil geralmente não possuem cadeia de frio, ou a possuem em temperatura entre 10 e 15ºC. Este trabalho teve como objetivo avaliar a qualidade e conservação do morango 'Oso Grande' sob temperatura de 10ºC associada com altas concentrações de dióxido de carbono. Os morangos foram selecionados, resfriados e armazenados a 10ºC em mini-câmaras herméticas, onde foram aplicadas as distintas concentrações de CO2 (0,03, 10, 20, 40 e 80%) combinadas com 20% de O2. Os morangos foram avaliados a cada 2 dias até se tornarem impróprios para o consumo. As concentrações de 20 e 40% de CO2 permitiram a conservação dos morangos por até 8 dias; já aqueles com 0,03% de CO2 duraram apenas 2 dias. Os morangos a 80% de CO2 mantiveram ótima aparência por 6 dias, porém foram considerados inadequados para o consumo por apresentarem elevados teores de acetaldeído (40,92 µg g-1) e de etanol (1.053 µg g-1), provenientes do processo fermentativo. A perda de massa fresca dos morangos foi inferior a 2%, demonstrando a eficiência da técnica utilizada para o controle da umidade relativa no armazenamento. Os frutos acondicionados com 0,03 e 80% de CO2 apresentaram a maior perda de firmeza, sendo que ao final do armazenamento esta foi de 40% em relação à firmeza inicial. Já os morangos armazenados com 20 e 40% de CO2 perderam apenas 28% da firmeza inicial. Apesar da diferença estatística na coloração externa do morango, essa foi visualmente imperceptível. Os morangos 'Oso Grande' armazenados a 10ºC sob atmosfera controlada com 40% de CO2 associado com 20% de O2 mantiveram suas características comerciais por 8 dias.

Characterization of Two Divergent Endo-β-1,4-Glucanase cDNA Clones Highly Expressed in the Nonclimacteric Strawberry Fruit

Two cDNAs clones (Cel1 and Cel2) encoding divergent endo-β-1,4-glucanases (EGases) have been isolated from a cDNA library obtained from ripe strawberry (Fragaria x ananassa Duch) fruit. The analysis of the amino acid sequence suggests that Cel1 and Cel2 EGases have different secondary and tertiary structures and that they differ in the presence of potential N-glycosylation sites. By in vitro translation we show that Cel1 and Cel2 bear a functional signal peptide, the cleavage of which yields mature proteins of 52 and 60 kD, respectively. Phylogenetic analysis revealed that the Cel2 EGase diverged early in evolution from other plant EGases. Northern analysis showed that both EGases are highly expressed in fruit and that they have different temporal patterns of accumulation. The Cel2 EGase was expressed in green fruit, accumulating as the fruit turned from green to white and remaining at an elevated, constant level throughout fruit ripening. In contrast, the Cel1 transcript was not detected in green fruit and only a low level of expression was observed in white fruit. The level of Cel1 mRNA increased gradually during ripening, reaching a maximum in fully ripe fruit. The high levels of Cel1 and Cel2 mRNA in ripe fruit and their overlapping patterns of expression suggest that these EGases play an important role in softening during ripening. In addition, the early expression of Cel2 in green fruit, well before significant softening begins, suggests that the product of this gene may also be involved in processes other than fruit softening, e.g. cell wall expansion.

Fruit cell culture as a model system to study cell wall changes during strawberry fruit ripening

Strawberry (Fragaria x ananassa, Duch.) fruit is characterized by its fast ripening and soft texture at the ripen stage, resulting in a short postharvest shelf life and high economic losses. It is generally believed that the disassembly of cell walls, the dissolution of the middle lamella and the reduction of cell turgor are the main factors determining the softening of fleshy fruits. In strawberry, several studies indicate that the solubilisation and depolymerisation of pectins, as well as the depolymerisation of xyloglucans, are the main processes occurring during ripening. Functional analyses of genes encoding pectinases such as polygalacturonase and pectate lyase also point out to the pectin fraction as a key factor involved in textural changes. All these studies have been performed with whole fruits, a complex organ containing different tissues that differ in their cell wall composition and undergo ripening at different rates. Cell cultures derived from fruits have been proposed as model systems for the study of several processes occurring during fruit ripening, such as the production of anthocyanin and its regulation by plant hormones. The main objective of this research was to obtain and characterize strawberry cell cultures to evaluate their potential use as a model for the study of the cell wall disassembly process associate with fruit ripening. Cell cultures were obtained from cortical tissue of strawberry fruits, cv. Chandler, at the stages of unripe-green, white and mature-red. Additionally, a cell culture line derived from strawberry leaves was obtained. All cultures were maintained in solid medium supplemented with 2.5 mg.l-1 2,4-D and incubated in the dark. Cell walls from the different callus lines were extracted and fractionated to obtain CDTA and sodium carbonate soluble pectin fractions, which represent polyuronides located in the middle lamella or the primary cell wall, respectively. The amounts of homogalacturonan in both fractions were estimated by ELISA using LM19 and LM20 antibodies, specific against demethylated and methyl-esterified homogalacturonan, respectively. In the CDTA fraction, the cell line from ripe fruit showed a significant lower amount of demethylated pectins than the rest of lines. By contrast, the content of methylated pectins was similar in green- and red-fruit lines, and lower than in white-fruit and leaf lines. In the sodium carbonate pectin fraction, the line from red fruit also showed the lowest amount of pectins. These preliminary results indicate that cell cultures obtained from fruits at different developmental stages differ in their cell wall composition and these differences resemble to some extent the changes that occur during strawberry softening. Experiments are in progress to further characterize cell wall extracts with monoclonal antibodies against other cell wall epitopes.

Unravelling the nanostructure of strawberry fruit pectins by atomic force microscopy

Atomic force microscopy (AFM) allows the analysis of individual polymers at nanostructural level with a minimal sample preparation. This technique has been used to analyse the pectin disassembly process during the ripening and postharvest storage of several fleshy fruits. In general, pectins analysed by AFM are usually visualized as isolated chains, unbranched or with a low number of branchs and, occasionally, as large aggregates. However, the exact nature of these structures is unknown. It has been suggested that pectin aggregates represent a mixture of rhamnonogalacturonan I and homogalacturonan, while isolated chains and their branches are mainly composed by polygalacturonic acid. In order to gain insight into the nature of these structures, sodium carbonate soluble pectins from ripe strawberry (Fragaria x ananassa, Duch.) fruits were subjected to enzymatic digestion with endo-Polygalacturonase M2 from Aspergillus aculeatus, and the samples visualized by AFM at different time intervals. Pectins isolated from control, non-transformed plants, and two transgenic genotypes with low level of expression of ripening-induced pectinase genes encoding a polygalacturonase (APG) or a pectate lyase (APEL) were also included in this study. Before digestion, isolated pectin chains from control were shorter than those from transgenic fruits, showing number-average (LN) contour length values of 73.2 nm vs. 95.9 nm and 91.4 nm in APG and APEL, respectively. The percentage of branched polymers was significantly higher in APG polyuronides than in the remaining genotypes, 33% in APG vs. 6% in control and APEL. As a result of the endo-PG treatment, a gradual decrease in the main backbone length of isolated chains was observed in the three samples. The minimum LN value was reached after 8 h of digestion, being similar in the three genotypes, 22 nm. By contrast, the branches were not visible after 1.5-2 h of digestion. LN values were plotted against digestion time and the data fitted to a first-order exponential decay curve, obtaining R2 values higher than 0.9. The half digestion time calculated with these equations were similar for control and APG pectins, 1.7 h, but significantly higher in APEL, 2.5 h, indicating that these polymer chains were more resistant to endo-PG digestion. Regarding the pectin aggregates, their volumes were estimated and used to calculate LN molecular weights. Before digestion, control and APEL samples showed complexes of similar molecular weights, 1722 kDa, and slightly higher than those observed in APG samples. After endo-PG digestion, size of complexes diminished significantly, reaching similar values in the three pectin samples, around 650 kDa. These results suggest that isolated polymer chains visualized by AFM are formed by a HG domain linked to a shorter polymer resistant to endo-PG digestion, maybe xylogalacturonan or RG-I. The silencing of the pectate lyase gene slightly modified the structure and/or chemical composition of polymer chains making these polyuronides more resistant to enzymatic degradation. Similarly, polygalacturonic acid is one of the main component of the aggregates.

Controle do crescimento de mudas de morangueiro 'Camarosa' cultivadas em substrato comercial pela aplicação de proexadione cálcio.

2016

Regulation of strawberry growth and development

Strawberries (Fragaria sp.) are adapted to diverse environmental conditions from the tropics to about 70ºN, so different responses to environmental conditions can be found. Most genotypes of garden strawberry (F. x ananassa Duch.) and woodland strawberry (F. vesca L.) are short-day (SD) plants that are induced to flowering by photoperiods under a critical limit, but also various photoperiod x temperature interactions can be found. In addition, continuously flowering everbearing (EB) genotypes are found. In addition to flowering, axillary bud differentiation in strawberry is regulated by photoperiod. In SD conditions, axillary buds differentiate to rosette-like structures called "branch crowns", whereas in long-day conditions (LD) they form runners, branches with 2 long internodes followed by a daughter plant (leaf rosette). The number of crown branches determines the yield of the plant, since inflorescences are formed from the apical meristems of the crown. Although axillary bud differentiation is an important developmental process in strawberries, its environmental and hormonal regulation has not been characterized in detail. Moreover, the genetic mechanisms underlying axillary bud differentiation and regulation of flowering time in these species are almost completely unresolved. These topics have been studied in this thesis in order to enhance strawberry research, cultivation and breeding. The results showed that 8-12 SD cycles suppressed runner initiation from the axillary buds of the garden strawberry cv. Korona with the concomitant induction of crown branching, and 3 weeks of SD was sufficient for the induction of flowering in the main crown. Furthermore, a second SD treatment given a few weeks after the first SD period can be used to induce flowering in the primary branch crowns and to induce the formation of secondary branches. Thus, artificial SD treatments effectively stimulate crown branching, providing one means for the increase of cropping (yield) potential in strawberry. It was also shown by growth regulation applications, quantitave hormone analysis and gene expression analysis that gibberellin (GA) is one of the key signals involved in the photoperiod control of shoot differentiation. The results indicate that photoperiod controls GA activity specifically in axillary buds, thereby determining bud fate. It was further shown that chemical control of GA biosynthesis by prohexadione-calcium can be utilized to prevent excessive runner formation and induce crown branching in strawberry fields. Moreover, ProCa increased berry yield up to 50%, showing that it is an easier and more applicable alternative to artificial SD treatments for controlling strawberry crown development and yield. Finally, flowering gene pathways in Fragaria were explored by searching for homologs of 118 Arabidopsis thaliana flowering-time genes. In total, 66 gene homologs were identified, and they distributed to all known flowering pathways, suggesting the presence of these pathways also in strawberry. Expression analysis of selected genes revealed that the mRNA of putative floral identity gene APETALA1 accumulated in the shoot apex of the EB genotype after the induction of flowering, whereas it was absent in vegetative SD genotype, indicating the usefulness of this gene product as the marker of floral initiation. The present data enables the further exploration of strawberry flowering pathways with genetic transformation, gene mapping and transcriptomics methods.

Characterization of an allotriploid strawberry Fragaria × bifera Duchesne (Rosaceae) from Europe

Peer reviewed

Targeted gene modification in Fragaria vesca mediated by CRISPR/Cas9 system

Genome editing is becoming an important biotechnological tool for gene function analysis and crop improvement, being the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein 9) system the most widely used. The natural CRISPR/Cas9 system has been reduced to two components: a single-guide RNA (sgRNA) for target recognition via RNA-DNA base pairing, which is commonly expressed using a promoter for small-RNAs (U6 promoter), and the Cas9 endonuclease for DNA cleavage (1). To validate the CRISPR/Cas9 system in strawberry plants, we designed two sgRNAs directed against the floral homeotic gene APETALA3 (sgRNA-AP3#1 and sgRNA-AP3#2). This gene was selected because ap3 mutations induce clear developmental phenotypes in which petals and stamens are missing or partially converted to sepals and carpels respectively (2). In this work, we used two different U6 promoters to drive the sgRNA-AP3s expression: AtU6-26 from Arabidopsis (4), and a U6 promoter from Fragaria vesca (FvU6) (this work). We also tested two different coding sequences of Cas9: a human- (hSpCas9) (3) and a plant-codon optimized (pSpCas9) (this work). Transient expression experiments using both CRISPR/Cas9 systems (AtU6-26:sgRNA-AP3#1_35S:hSpCas9_AtU6-26:sgRNA-AP3#2 and FvU6:sgRNA-AP3#1_35S:pSpCas9_FvU6:sgRNA-AP3#2) were performed infiltrating Agrobacterium tumefaciens into F. vesca fruits. PCR amplification and sequencing analyses across the target sites showed a deletion of 188-189 bp corresponding to the region comprised between the two cutting sites of Cas9, confirming that the CRISPR/Cas9 system is functional in F. vesca. Remarkably, the two systems showed different mutagenic efficiency that could be related to differences in expression of the U6 promoters as well as differences in the Cas9 transcripts stability and translation. Stable transformants for both F. vesca (2n) and Fragaria X anannassa (8n) are currently being established to test whether is possible to obtain heritable homozygous mutants derived from CRISPR/Cas9 strategies in strawberry. Thus, our work offers a promising tool for genome editing and gene functional analysis in strawberry. This tool might represent a more efficient alternative to the sometimes inefficient RNAi silencing methods commonly used in this species.

Bases nutricionales del cultivo de frutilla (Fragaria ananassa Duch.)

El sistema de producción invernal de frutilla, dominado por 'Camarosa'., tiende a ampliar su espectro varietal. Las recomendaciones actuales de fertilización no consideran particularidades genéticas ni eficiencia en el uso de los nutrientes (EUNu). Los objetivos de este trabajo de tesis son: estudiar la respuesta de cultivares de frutilla contrastantes en morfología y fisiología a incrementos en la fertilización NPKCaMg, determinar e interpretar índices de EUNu y cuantificar efectos ambientales, genotípicos y nutricionales sobre los atributos de calidad de frutos. Se evaluaron 'Camarosa'., 'Camino Real', 'Sabrosa' y 'Ventana' ante incrementos en la fertilización, a partir de un tratamiento testigo '1' (120N, 30P, 186K, 31Ca y 12Mg kg/ha) proporcionalmente a '1,5' en 2006, '1,33' y '1,66' en 2007, '1,5' y '2' en 2008. Esto determinó una respuesta genotípica diferencial cuadrática que definió dosis óptimas: 'Camarosa' 15 por ciento a dosis 1,53, 'Camino Real' 6 por ciento a dosis 1,36, 'Sabrosa' 4 por ciento a dosis 1,29 y 'Ventana' 19 por ciento a dosis 1,59. A mitad del ciclo, el número de hojas se correlacionó con el rendimiento total y los cultivares de mayor respuesta mostraron mayor K y menor Ca foliar, acentuado al aumentar la fertilización. La productividad del nutriente aportado (rendimiento/fertilización) resultó mayor en cultivares más productivos y de mayor respuesta. El balance de nutrientes (absorción/fertilización) dependió de la interacción genotipo por ambiente. La eficiencia de recuperación (absorción/fertilización) fue proporcional a la respuesta a la fertilización. Los coeficientes de eficiencia interna (biomasa/absorción y rendimiento/absorción) resultaron diferentes entre cultivares y consistentes entre tratamientos de fertilización. Peso de fruto y sólidos solubles totales variaron con la interacción genotipo por ambiente; firmeza, tono de color, y acidez con el genotipo y el ambiente; ninguno de ellos varió la nutrición. Los resultados sientan bases nutricionales para el cultivo de frutilla, en cuanto a la absorción genotípico-diferencial de nutrientes y sus efectos en el crecimiento, producción y EUNu.