Flavonoids are secondary metabolites that have significant roles in plant defense and human nutrition. Glucosyltransferases (GTs) transfer sugars from high energy sugar donors to other substrates. Several different kinds of flavonoid GTs exist in the tissues of grapefruit making it a model plant for studying their structure and function. The goal of this investigation is to determine the expression patterns of 7 putative secondary product GTs during grapefruit growth and development by quantifying mRNA expression levels in the roots, stems, leaves, and flowers. This research was designed to test the hypothesis that these 7 GT's are expressed constitutively. Alternatively, one or more could be expressed in a tissue-specific manner and/or developmentally regulated. Six growth stages were defined. Findings show that there were variable degrees of PGT expression. Therefore, results were more consistent with the alternative hypothesis that putative secondary product GT expression was tissue specific and/or developmentally regulated.

Em dois agroecossistemas distintos (Piracicaba, SP, Brasil e Okeechobee, FL, EUA) cultivados com citros objetivou-se avaliar: i) a concentração de nutrientes, metais pesados no efluente de esgoto tratado (EET) e sua qualidade para o uso agrícola, e; ii) os efeitos da irrigação com EET na fertilidade do solo e nutrição das plantas. Em Piracicaba, SP, foi conduzido um experimento com três blocos casualizados e cinco tratamentos. Quatro lâminas de irrigação distintas com EET foram aplicadas durante 21 meses em laranjeiras [Citrus sinensis (L.) Osb.] sobre Citrus paradisi Macfad. x Poncirus trifoliata Raf] com base na evapotranspiração da cultura (ETc) sendo: 100%, 125%, 150% e 200% da ETc, além do tratamento sem irrigação (0%). Em Okeechobee, FL, foi realizado um estudo de caso em um pomar comercial cultivado com pomeleiros [(Citrus paradise Macf.) sobre (Citrus aurantium L.)]. Neste local comparou-se dois talhões; um irrigado com EET (durante 11 anos) e outro irrigado com água. Os EETs utilizados nos dois estudos apresentaram qualidade satisfatória para o uso agrícola (FAO, 1992). No primeiro agroecossistema houve correlação negativa entre as lâminas de irrigação com EET e a acidez ativa, acidez potencial, saturação por alumínio e os teores de K trocável e Pb disponível. Por outro lado, correlações positivas foram encontradas entre S, B, Cu e Na no solo e as lâminas de irrigação. Comparando-se a lâmina de irrigação 100% da ETc e o controle (0%), houve um aumento na abundância natural de 15N e nas concentrações de S, B e Na, e um decréscimo de P, K, Mn, Al e Cr nas folhas. As menores concentrações destes elementos foram associadas às lâminas excessivas (> 100% da ETc). A influência máxima das lâminas de irrigação com EET na altura das plantas foi obtida entre as lâminas 93 e 107% da ETc. Quanto ao segundo agroecossistema, houve um aumento na concentração de NH4 +, NO3 -, Mg, S, B, Cl, Co, Cu, Fe, Mn, Mo, Ni e Zn disponíveis no solo após 11 anos de irrigação com EET. Já no tecido foliar houve um acréscimo de Ca, Mg, B, Fe, Cu, Mn, Ni, e Zn. As concentrações de Cd, Co, Mo e Pb foram encontradas abaixo do limite de detecção no tecido foliar. O efeito do EET é pronunciado no longo prazo, e pode suprir parcialmente alguns nutrientes do programa de adubação. Mesmo após longo período de irrigação com EET, o risco de salinização do solo e acúmulo de metais pesados parecem mínimos. Com base nestes resultados fica evidente que além de ser uma fonte alternativa de água, o EET influencia positivamente na fertilidade do solo e nutrição dos citros. / Considering two different agroecosystems (Piracicaba, SP, Brazil and Okeechobee, FL, USA) cultivated with citrus, the objectives of the study were to evaluate: i) the concentration of nutrients, heavy metals in reclaimed wastewater (RWW) and its quality for the agricultural use; and ii) the effects of RWW irrigation on soil fertility and plant nutrition. At Piracicaba, SP, an experiment were carried out with three randomized blocks and five treatments. Four different RWW irrigation rates were applied over 21 months, in orange trees [Citrus sinensis (L.) Osb] on Citrus paradisi Macfad. x Poncirus trifoliata Raf], based on crop evapotranspiration (ETc): 100%, 125%, 150% and 200% of ETc as well as a treatment without irrigation (0%). At Okeechobee, FL, a survey study was carried out in a commercial citrus grove cultivated with grapefruit [(Citrus paradise Macf.) on (Citrus aurantium L.)]. At this site two citrus blocks were compared; one irrigated with RWW (11 years) and the other with well water. The RWW used in both studies displayed satisfactory quality for the agricultural use (FAO, 1992). The first agroecosystem had negative correlation between RWW irrigation rates and active acidity, potential acidity, aluminum saturation, exchangeable K and available Pb. Whereas positive correlations were found for S, B, Cu and Na in soil with RWW rates. Comparing the irrigation rate of 100% of ETc with the control plot (0%), increasing contents of N, 15N, S, B, and decreasing contents of P, K, Mn, Al and Cr were observed in the leaf tissue. Lower concentrations of these elements were associated with the over irrigation rates (> 100% of ETc). The greatest effect of the irrigation rates on plants height was found between 93 and 107% of ETc. At the second agroecosystem, increasing concentration of NH4 +, NO3 -, Mg, S, B, Cl, Co, Cu, Fe, Mn, Mo, Ni and Zn in soil were found after 11 years of RWW irrigation. In leaf tissue also increasing concentrations of Ca, Mg, B, Fe, Cu, Mn, Ni and Zn were determined. Concentration of Cd, Co, Mo and Pb were below the detection limit of detection in the leaf tissue. The effects of the RWW irrigation are pronounced after long term application, and can partially supply some nutrients from the fertilization program. Even after long term RWW irrigation, the risks of soil salinization and heavy metals accumulation can be considered as low. The results highlight that besides an alternative waster source, RWW can provide benefits on soil fertility and citrus nutrition.

Em dois agroecossistemas distintos (Piracicaba, SP, Brasil e Okeechobee, FL, EUA) cultivados com citros objetivou-se avaliar: i) a concentração de nutrientes, metais pesados no efluente de esgoto tratado (EET) e sua qualidade para o uso agrícola, e; ii) os efeitos da irrigação com EET na fertilidade do solo e nutrição das plantas. Em Piracicaba, SP, foi conduzido um experimento com três blocos casualizados e cinco tratamentos. Quatro lâminas de irrigação distintas com EET foram aplicadas durante 21 meses em laranjeiras [Citrus sinensis (L.) Osb.] sobre Citrus paradisi Macfad. x Poncirus trifoliata Raf] com base na evapotranspiração da cultura (ETc) sendo: 100%, 125%, 150% e 200% da ETc, além do tratamento sem irrigação (0%). Em Okeechobee, FL, foi realizado um estudo de caso em um pomar comercial cultivado com pomeleiros [(Citrus paradise Macf.) sobre (Citrus aurantium L.)]. Neste local comparou-se dois talhões; um irrigado com EET (durante 11 anos) e outro irrigado com água. Os EETs utilizados nos dois estudos apresentaram qualidade satisfatória para o uso agrícola (FAO, 1992). No primeiro agroecossistema houve correlação negativa entre as lâminas de irrigação com EET e a acidez ativa, acidez potencial, saturação por alumínio e os teores de K trocável e Pb disponível. Por outro lado, correlações positivas foram encontradas entre S, B, Cu e Na no solo e as lâminas de irrigação. Comparando-se a lâmina de irrigação 100% da ETc e o controle (0%), houve um aumento na abundância natural de 15N e nas concentrações de S, B e Na, e um decréscimo de P, K, Mn, Al e Cr nas folhas. As menores concentrações destes elementos foram associadas às lâminas excessivas (> 100% da ETc). A influência máxima das lâminas de irrigação com EET na altura das plantas foi obtida entre as lâminas 93 e 107% da ETc. Quanto ao segundo agroecossistema, houve um aumento na concentração de NH4 +, NO3 -, Mg, S, B, Cl, Co, Cu, Fe, Mn, Mo, Ni e Zn disponíveis no solo após 11 anos de irrigação com EET. Já no tecido foliar houve um acréscimo de Ca, Mg, B, Fe, Cu, Mn, Ni, e Zn. As concentrações de Cd, Co, Mo e Pb foram encontradas abaixo do limite de detecção no tecido foliar. O efeito do EET é pronunciado no longo prazo, e pode suprir parcialmente alguns nutrientes do programa de adubação. Mesmo após longo período de irrigação com EET, o risco de salinização do solo e acúmulo de metais pesados parecem mínimos. Com base nestes resultados fica evidente que além de ser uma fonte alternativa de água, o EET influencia positivamente na fertilidade do solo e nutrição dos citros. / Considering two different agroecosystems (Piracicaba, SP, Brazil and Okeechobee, FL, USA) cultivated with citrus, the objectives of the study were to evaluate: i) the concentration of nutrients, heavy metals in reclaimed wastewater (RWW) and its quality for the agricultural use; and ii) the effects of RWW irrigation on soil fertility and plant nutrition. At Piracicaba, SP, an experiment were carried out with three randomized blocks and five treatments. Four different RWW irrigation rates were applied over 21 months, in orange trees [Citrus sinensis (L.) Osb] on Citrus paradisi Macfad. x Poncirus trifoliata Raf], based on crop evapotranspiration (ETc): 100%, 125%, 150% and 200% of ETc as well as a treatment without irrigation (0%). At Okeechobee, FL, a survey study was carried out in a commercial citrus grove cultivated with grapefruit [(Citrus paradise Macf.) on (Citrus aurantium L.)]. At this site two citrus blocks were compared; one irrigated with RWW (11 years) and the other with well water. The RWW used in both studies displayed satisfactory quality for the agricultural use (FAO, 1992). The first agroecosystem had negative correlation between RWW irrigation rates and active acidity, potential acidity, aluminum saturation, exchangeable K and available Pb. Whereas positive correlations were found for S, B, Cu and Na in soil with RWW rates. Comparing the irrigation rate of 100% of ETc with the control plot (0%), increasing contents of N, 15N, S, B, and decreasing contents of P, K, Mn, Al and Cr were observed in the leaf tissue. Lower concentrations of these elements were associated with the over irrigation rates (> 100% of ETc). The greatest effect of the irrigation rates on plants height was found between 93 and 107% of ETc. At the second agroecosystem, increasing concentration of NH4 +, NO3 -, Mg, S, B, Cl, Co, Cu, Fe, Mn, Mo, Ni and Zn in soil were found after 11 years of RWW irrigation. In leaf tissue also increasing concentrations of Ca, Mg, B, Fe, Cu, Mn, Ni and Zn were determined. Concentration of Cd, Co, Mo and Pb were below the detection limit of detection in the leaf tissue. The effects of the RWW irrigation are pronounced after long term application, and can partially supply some nutrients from the fertilization program. Even after long term RWW irrigation, the risks of soil salinization and heavy metals accumulation can be considered as low. The results highlight that besides an alternative waster source, RWW can provide benefits on soil fertility and citrus nutrition.

Flavonoids are secondary metabolites that are important in plant defense, protection, and human health. Most naturally-occurring flavonoids are found in glucosylated forms. Glucosyltransferases catalyze the transfer of glucose from high-energy sugar donors to an acceptor molecule. The grapefruit flavonol-specific 3-O-glucosyltransferase (F3-O-GT) is highly substrate and regio-specific. The goal of this research is to unravel the amino acid residues responsible for the grapefruit enzyme’s rigid specificity, while attempting to alter the regiospecific glucosylation pattern through site-directed mutagenesis and homology modeling. This research tested the hypothesis that substitution of potential key amino acid residues within the grapefruit Cp-F3-O-GT with position equivalent residues within F7-O-GTs would alter the 3-O-glucosylation of the enzyme. Results reveal that specific single point mutations of residues are capable of abolishing enzymatic activity. Recombinant mutant G392E retained activity and showed an increased affinity for kaempferol relative to the wild-type; however, the rigid regiospecific glucosylation pattern of the enzyme was retained.

The wide diversity of plant secondary products results from different modifications undergone during biosynthesis, including glucosylation. These modification reactions result in production of the compounds actually found in plants and to unique chemical and biochemical properties, including some bitter compounds in grapefruit. While the presence of a PSPG box motif allows for identification of a clone as a putative glucosyltransferase (PGT), diversity of GT primary structures makes it difficult to accurately assign specific function. Our approach is to identify and isolate putative GT clones, express them heterologously, and biochemically characterize the proteins. Eleven putative GT clones have been isolated from Citrus paradise and some have been biochemically characterized. The current hypothesis being tested is that PGT3 and PGT9 clones are plant secondary product GTs. Due to issues with inclusion bodies when using E. coli, proteins were expressed in Pichia pastoris using the pPICZA vector. Recombinant protein expression was confirmed by Western blot and proteins were enriched by IMAC. Over 30 flavonoid and simple phenolic substrates, representing many compounds found in grapefruit, were screened for activity with PGT3 and PGT9 proteins. No significant activity was found and the biochemical function of the proteins encoded by these clones will be further investigated.

Glucosyltransferases (GTs) are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi has strict substrate and regiospecificity (Cp3OGT). The amino acid sequence of Cp3OGT was aligned with a purported anthocyanin GT from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling to identify candidate regions followed by site directed mutagenesis, three double mutations of Cp3OGT were made. Biochemical analysis of the three mutant proteins was performed. S20G+T21S protein retained activity similar to the wildtype (WT- Kmapp-80 µM; Vmax = 16.5 pkat/µg, Mutant- Kmapp-83 µM; Vmax -11 pkat/µg) but the mutant was more thermostable compared to the WT and this mutation broadened its substrate acceptance to include the flavanone, naringenin. S290C+S319A mutant protein retained 40% activity relative to wildtype, had an optimum pH shift, but had no change in substrate specificity (Kmapp-18 µM; Vmax-0.5 pkat/µg). H154Y+Q87I protein was inactive with every class of flavonoid tested. Product identification revealed that the S20G+T21S mutant protein widened the substrate and regio-specificity of CP3OGT. Docking analysis revealed that H154 and Q87 could be involved in orienting the ligand molecules within the acceptor binding site. H363, S20, and S150 were also found to make close contact with the 7-OH, 4-OH and 3’-OH groups, respectively.

No description available.

Recombinant tags are used extensively in protein expression systems to allow purification through IMAC (Immobilized Metal Affinity Chromatography), identification through Western blot, and to facilitate crystal formation for structural analysis. While widely used, their role in enzyme characterization has raised concerns with respect to potential impact on activity. In this study, a flavonol-specific 3-O glucosyltransferase (Cp3GT) from grapefruit (Citrus paradisi) was expressed in Pichia pastoris, and was assayed in its untagged form and with a C-terminal c-myc/6x His tag under various conditions to determine the effect of tags. Prior characterization of pH optima for Cp3GT obtained through expression in Escherichia coli, containing an N-terminal thioredoxin/6x His tag, indicated an optimal pH of 7–7.5, which is indicative of a normal physiological pH and agrees with other glucosyltransferase (GT) pH optima. However, characterization of Cp3GT expressed using P. pastoris with a C-terminal c-myc-6x His tag showed a higher optimal pH of 8.5–9. This suggests a possible tag effect or an effect related to physiological differences between the cell expression systems. Results testing recombinant Cp3GT expressed in Pichia with and without C-terminal tags showed a possible tag effect with regard to substrate preference and interactions with metals, but no apparent effect on enzymatic kinetics or pH optima.

O crescente aumento da tolerância aos fungicidas atualmente utilizados é um dos principais problemas tanto na área clínica quanto na agrícola e tem estimulado o desenvolvimento de estratégias alternativas para o controle de fungos patogênicos, tais como o tratamento fotodinâmico antimicrobiano (TFA), que pode utilizar fotossensibilizadores (FS) de origem natural ou sintética. Alguns FS, como as cumarinas e furanocumarinas, são produzidos por espécies de Citrus como limão Tahiti (Citrus latifolia) e toranjas (Citrus paradisi). O presente estudo investigou o TFA in vitro de conídios do fungo fitopatogênico Colletotrichum acutatum com: (1) o corante fenotiazínico azul de metileno (MB), (2) uma furanocumarina comercial, 8-metoxipsoraleno (8-MOP), (3) uma cumarina comercial, (2H-chromen-2-one), (4) a cumarina 5,7-dimetoxicumarina extraída no presente trabalho a partir de óleo da casca do limão Tahiti, (5) a cumarina 7-hidroxicumarina extraída no presente trabalho a partir de óleo essencial de toranja rubi, (6) duas frações extraídas por De Menezes e colaboradores (2014a) a partir de um resíduo da indústria citrícola, uma delas constituída pela furanocumarina isopimpinelina e outra constituída por uma mistura das cumarinas 7-metoxicumarina e 5,7-dimetoxicumarina (3:1), (7) cinco extratos brutos obtidos a partir de óleos da casca e essencial de limão Tahiti e óleos essenciais de toranjas rubi e branca. Com exceção do extrato bruto obtido do óleo essencial de toranja branca, todos os demais extratos apresentaram cumarinas e/ou furanocumarinas em sua composição. As suspensões de conídios foram tratadas com os diferentes compostos (50 ?M para os compostos comerciais e as frações puras e 12,5 mg L-1 para os extratos e a mistura) e expostas à radiação solar por 1 h. O MB foi exposto à radiação solar por 30 min, 1 e 2 h. Determinou-se o efeito do tratamento fotodinâmico em folhas de mudas de laranja Valencia (Citrus sinensis) com (1) 8-MOP, (2) mistura de cumarinas, (3) 5,7-dimetoxicumarina e (4) o extrato bruto de limão Tahiti. Adicionalmente, experimentos para avaliar a estabilidade do 8-MOP exposto à radiação solar por até 12 h foram realizados. Nesses experimentos, soluções de 8-MOP (50 ?M) foram expostas à radiação solar e alíquotas foram retiradas após os diferentes tempos de exposição. Após as exposições, estudos de espectroscopia de absorção no UV e emissão de fluorescência foram realizados, além da realização de experimentos para avaliar a eficácia do TFA com os FS previamente expostos à radiação solar por 6 e 12 h. Os TFA mais eficazes foram com o MB, 8-MOP (ambos 50 ?M), a mistura de cumarinas e um dos extratos brutos obtidos de limão Tahiti (ambos 12,5 mg L-1), que mataram 99,998%, 99,378%, 99,336% e 65,049% dos conídios, respectivamente, após 1 h de exposição à radiação solar. Nenhum dano nas folhas de laranja foi observado após o TFA com os compostos. Os espectros de absorção e emissão de fluorescência das soluções de 8-MOP expostas à radiação solar mostraram fotodegradação do composto após 12 h de exposição. Entretanto, as soluções de 8-MOP expostas por 6 e por 12 h ainda foram capazes de matar 99,978% e 95,591% dos conídios de C. acutatum, respectivamente / Increasing tolerance to currently used fungicides is a major problem both in clinical and agricultural areas and has stimulated the development of alternative strategies to control pathogenic fungi such as the antimicrobial photodynamic treatment (APDT), that can use natural or synthetic photosensitizers (PS). Some PS, such as coumarins and furocoumarins are produced by Citrus species such Tahiti acid lime (Citrus latifolia) and grapefruit (Citrus paradisi). This study investigated the in vitro APDT of conidia of the plant-pathogenic fungi C. acutatum with: (1) the phenothiazinium dye methylene blue (MB), (2) commercial furocoumarin, 8-methoxypsoralen (8-MOP), (3) commercial coumarin (2H-chromen-2-one), (4) coumarin 5,7-dimethoxycoumarin, extracted in this study from Tahiti lime peel oil, (5) coumarin 7-hidroxycoumarina, extracted in this study from ruby grapefruit essential oil, (6) two fractions extracted by De Menezes et al (2014a) from a Citrus industry residue, one of them constituted by the furocoumarin isopimpinellin, and the other constituted by the mixture of coumarins 7-methoxycoumarin and 5,7-dimethoxycoumarin (3:1), (7) five crude extracts obtained from Tahiti lime peel or essential oil and ruby and white grapefruit essential oil. Except the extract from the white grapefruit, all the others presented coumarins and/or furocoumarins in their composition. Conidial suspentions were treated with the different PS (50 ?M for commercial and pure compounds and 12.5 mg L-1 for the extracts and the mixture) and exposed to solar radiation for 1 h. The MB was exposed to solar radiation for 30 min, 1 and 2 h. The APDT effect on the leaves of Valencia orange trees (Citrus sinensis) with (1) 8-MOP, (2) the mixture of coumarins, (3) 5,7-dimethoxycoumarin, and (4) the most efective extract from Tahiti lime were determined. Additionally, experiments to assess the 8-MOP stability after exposure to solar radiation for 12 h were realized. 8-MOP solutions (50 ?M) were exposed under solar radiation and rates were removed after different times of exposition. After the expositions, absorption spectroscopic studies in UV and fluorescence emission were realized, as well as holding the realization of experiments of APDT efficacy evaluation after 6 and 12 h to exposure under solar radiation. The more effective treatments were with MB, 8-MOP (both 50 ?M), the coumarins mixture and one of the extracts from Tahiti lime (both 12.5 mg L-1), killing at least 99,998%, 99,378%, 99,336% and 65,049% of the conidia, respectively after 1 h under solar radiation. No damage to orange tree leaves was observed after APDT with any of the compounds. The absorption and fluorescence intensity spectra showed the 8-MOP photodegradation after 12 h of exposition, although the 8-MOP solutions exposed under solar radiation for 6 and 12 h still killed at least 99,978% and 95,591% of the C. acutatum conidia, respectively

Flavonoids are secondary metabolites that are important in plant defense, protection, and human health. Most naturally-occurring flavonoids are found in glucosylated form. Glucosyltransferases (GTs) are enzymes that catalyze the transfer of glucose from a high energy sugar donor to an acceptor molecule. At this time, it is not possible to accurately predict putative GT activity from sequence alone; biochemical characterization is critical. A flavonol-specific 3-O-GT enzyme has been identified and cloned from the leaf tissues of grapefruit. The enzyme shows rigid substrate specificity and regiospecificity. F3GTs from grape and grapefruit were modeled against F7GTs from Crocus sativus and Scrutellaria biacalensis, and several non-conservative amino acid differences were identified that may impact regioselectivity. This research is designed to test the hypothesis that specific amino acid residues impart the regiospecificity of the grapefruit enzyme. Site-directed mutagenesis was performed on three potentially key amino acid residues within the grapefruit F3-GT that were identified through homology modeling. Enzyme activity of the mutant F3-GT proteins will be analyzed for a possible change in glucosylation pattern. Other flavonoid classes will also be tested with the mutant enzymes to test for change in substrate specificity.