ABSTRACT Cell suspension culture has been used as a model to study metabolic changes to several stresses. To have detailed information of nitrogen (N) limitation on sugarcane metabolism, a controlled study of the primary metabolites and representative compounds of secondary metabolism was developed using suspension cells growing under three different N regimes: normal condition (40 mmol·L–1 NO-3), slightly deficient (12 mmol·L–1 NO-3) and completely deficient (0 mmol·L–1 NO-3). Sugarcane cells were harvested after 3 and 7 days of treatment. A range of changes in the levels of amino acids, organic acids, sugars and phenolic compounds were observed upon the growth conditions applied. Nitrogen limitation remarkably affected the amino acids and carbohydrates biosynthesis, which, associated with the changes observed on phenolic compounds contents, indicates the upregulation of carbon sink compensation mechanisms in these sugarcane cells exposed to N starvation. As expected, the results showed that N limitation might cause an extensive metabolic reprogramming of both carbon and N metabolism in sugarcane cells, and these changes are related to the intensity of the starvation. Nitrogen is essential for plant growth and development, and its limitation sharply reduces crop yield. Thus, these results open new perspectives for in planta studies concerning carbon and N metabolisms balance in this crop.

ABSTRACT Saccharum spontaneum has been used for the development of energy cane a crop aimed to be used for the production of second-generation ethanol, or lignocellulosic ethanol. Lignin is a main challenge in the conversion of cell wall sugars into ethanol. In our studies to isolate the genes the lignin biosynthesis in S. spontaneum we have had great difficulty in RT-PCR reactions. Thus, we evaluated the effectiveness of different additives in the amplification of these genes. While COMT and CCoAOMT genes did not need any additives for other genes there was no amplification (HCT, F5H, 4CL and CCR) or the yield was very low (CAD and C4H). The application of supplementary cDNA was enough to overcome the non-specificity and low yield for C4H and C3H, while the addition of 0.04% BSA + 2% formamide was effective to amplify 4CL, CCR, F5H and CCR. HCT was amplified only by addition of 0.04% BSA + 2% formamide + 0.1 M trehalose and amplification of PAL was possible with addition of 2% of DMSO. Besides optimization of expression assays, the results show that additives can act independently or synergistically.

Eucalyptus urograndis is a hybrid eucalyptus of major economic importance to the Brazilian pulp and paper industry. Although widely used in forest nurseries around the country, little is known about the biochemical changes imposed by environmental stress in this species. In this study, we evaluated the changes in the stem proteome after short-term stimulation by exposure to low temperature. Using two-dimensional gel electrophoresis coupled to high-resolution mass spectrometry-based protein identification, 12 proteins were found to be differentially regulated and successfully identified after stringent database searches against a protein database from a closely related species (Eucalyptus grandis). The identification of these proteins indicated that the E. urograndis stem proteome responded quickly to low temperature, mostly by down-regulating specific proteins involved in energy metabolism, protein synthesis and signaling. The results of this study represent the first step in understanding the molecular and biochemical responses of E. urograndis to thermal stress.

Nutrient analysis is used to estimate nutrient content of crop plants to manage fertilizer application for sustained crop production. Direct solid analysis of agricultural and environmental samples by energy dispersive X-ray fluorescence spectrometry (EDXRF) was chosen as alternative technique to evaluate the simultaneous multielemental quantification of the most important essential elements in coffee (Coffea arabica L.) plants. Inductively coupled plasma atomic emission spectrometry and certified reference materials made from leaves were used to calibrate and check the trueness of EDXRF method for the determination of the concentration of several nutrients in coffee leaves and branches. Fluorescence spectrometry proved to be advantageous and presented low cost as loose powder samples could be used. Samples collected from a field experiment where coffee plants were treated with excess of Ni and Zn were used to verify the practical application of the method. Good relationships were achieved between certified values and data obtained by EDXRF, with recoveries ranging from 82 to 117 %.

Six aquaporin (AQP) homologues were identified in three coffee species (Coffea arabica, C. Canephora, and C. racemosa), including four plasma membrane intrinsic proteins (PIP1;1, PIP1;2, PIP2;1, and PIP2;2) and two tonoplast intrinsic proteins (TIP1;1 and TIP1;2). In order to better understand the role of these genes in coffee, C. arabica cvs Catuaí and Mundo Novo, C. canephora cv. Apoatã, and a graft of Mundo Novo on Apoatã were water stressed, and the expression levels of PIP1;2, PIP2;1, PIP2;2, and TIP1;2 were analyzed in the roots and leaves. The expression of PIP2;1 and PIP2;2 was clearly up-regulated in the leaves and roots by water deficit, suggesting the possible involvement of these genes in controlling the water status of plants and in the post-stress recovery of irrigated plants. The most strongly induced expression in roots was found in both grafted and non-grafted Apoatã plants. Interestingly, the level of PIP2;1 transcripts in roots continued to rise even after the plants were watered and were, therefore, no longer subjected to the water stress, suggesting that this gene may be actively involved in the regulation of water uptake in coffee tree roots, especially when there is a water deficit in the soil.

Foram estudadas a atividade de enzimas antioxidantes e a expressão da ferritina em culturas celulares de duas variedades de Coffea arabica, Mundo Novo e Icatu, tratadas com alumínio (Al) em meio de cultura com pH 5.8. As células foram incubadas com 300 µM Al3+, e a especiação do Al para Al3+ foi de 1.45%. Para Mundo Novo, a atividade da superóxido dismutase (SOD), catalase (CAT) e glutationa S-transferase (GST) aumentou, enquanto que a atividade da glutationa redutase (GR) e da guaiacol peroxidase (GPOX) permaneceu inalterada. Para Icatu, a atividade da SOD, GR e GST aumentou, a atividade da CAT não se alterou e da GPOX diminuiu. Foram analisadas a expressão de dois transcritos da ferritina (CaFer1 e CaFer2) por meio de PCR em Tempo Real. O Al provocou queda na expressão do CaFER1 e nenhuma alteração em CaFER2 nas duas variedades. Não houve alteração no acúmulo de ferritina para Mundo Novo, mas uma diminuição para Icatu. A resposta diferencial das enzimas indica que a resposta antioxidante é variedade dependente.

The effects of aluminum (Al) on the activities of antioxidant enzymes and ferritin expression were studied in cell suspension cultures of two varieties of Coffea arabica, Mundo Novo and Icatu, in medium with pH at 5.8. The cells were incubated with 300 µM Al3+, and the Al speciation as Al3+ was 1.45% of the mole fraction. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) were increased in Mundo Novo, whereas glutathione reductase (GR) and guaiacol peroxidase (GPOX) activities remained unchanged. SOD, GR, and GST activities were increased in Icatu, while CAT activity was not changed, and GPOX activity decreased. The expression of two ferritin genes (CaFer1 and CaFer2) were analyzed by Real-Time PCR. Al caused a downregulation of CaFER1 expression and no changes of CaFER2 expression in both varieties. The Western blot showed no alteration in ferritin protein levels in Mundo Novo and a decrease in Icatu. The differential enzymes responses indicate that the response to Al is variety-dependent.

Foram comparadas as características bioquímicas das sementes de um cafeeiro Arabica naturalmente descafeinado (AC1), descoberto em 2004, com aquelas da cultivar Mundo Novo (MN), amplamente cultivada no Brasil. Apesar de terem sido observadas diferenças durante o desenvolvimento das sementes, os conteúdos de aminoácidos, ácidos orgânicos, ácidos clorogênicos, açúcares solúveis e trigonelina foram similares nas sementes de frutos maduros de AC1 e MN. Sementes de AC1 acumularam teobromina, e a cafeína estava praticamente ausente. Experimentos com o fornecimento de [2-14C] adenina e análises enzimáticas de teobromina sintase e cafeína sintase nas sementes de AC1 confirmaram que, assim como em folhas, a síntese de cafeína é bloqueada na metilação de teobromina a cafeína. A qualidade final da bebida de AC1 foi similar a de MN.

We compared the biochemical characteristics of the beans of a naturally decaffeinated Arabica coffee (AC1) discovered in 2004 with those of the widely grown Brazilian Arabica cultivar "Mundo Novo" (MN). Although we observed differences during fruit development, the contents of amino acids, organic acids, chlorogenic acids, soluble sugars and trigonelline were similar in the ripe fruits of AC1 and MN. AC1 beans accumulated theobromine, and caffeine was almost entirely absent. Tests on the supply of [2-14C] adenine and enzymatic analysis of theobromine synthase and caffeine synthase in the endosperm of AC1 confirmed that, as in the leaves, caffeine synthesis is blocked during the methylation of theobromine to caffeine. The quality of the final coffee beverage obtained from AC1 was similar to that of MN.

Lignin, after cellulose, is the second most abundant biopolymer on Earth, accounting for 30% of the organic carbon in the biosphere. It is considered an important evolutionary adaptation of plants during their transition from the aquatic environment to land, since it bestowed the early tracheophytes with physical support to stand upright and enabled long-distance transport of water and solutes by waterproofing the vascular tissue. Although essential for plant growth and development, lignin is the major plant cell wall component responsible for biomass recalcitrance to industrial processing. The fact that lignin is a non-linear aromatic polymer built with chemically diverse and poorly reactive linkages and a variety of monomer units precludes the ability of any single enzyme to properly recognize and degrade it. Consequently, the use of lignocellulosic feedstock as a renewable and sustainable resource for the production of biofuels and bio-based materials will depend on the identification and characterization of the factors that determine plant biomass recalcitrance, especially the highly complex phenolic polymer lignin. Here, we summarize the current knowledge regarding lignin metabolism in plants, its effect on biomass recalcitrance and the emergent strategies to modify biomass recalcitrance through metabolic engineering of the lignin pathway. In addition, the potential use of sugarcane as a second-generation biofuel crop and the advances in lignin-related studies in sugarcane are discussed.

Fruits were collected from trees of Coffea arabica cv. Obatã grown at Mococa and Adamantina in São Paulo State, Brazil, which are regions with marked differences in air temperature that produce coffee with distinct qualities. Mococa is a cooler location that produces high-quality coffee, whereas coffee from Adamantina is of lower quality. The amino acid and protein contents, amino acid profile, and proteinase activity and type in endosperm protein extracts were analysed. Proteinase genes were identified, and their expression was assayed. All results indicate that temperature plays a role in controlling proteinase activity in coffee endosperm. Proteinase activity was higher in the endosperm of immature fruits from Adamantina, which was correlated with higher amino acid content, changes in the amino acid profile, and increased gene expression. Cysteine proteinases were the main class of proteinases in the protein extracts. These data suggest that temperature plays an important role in coffee quality by altering nitrogen compound composition.

A produtividade das plantas e a capacidade de resistência à seca estão diretamente relacionadas com a eficiência da condutância hidráulica do xilema e a capacidade desse sistema em evitar a interrupção do fluxo de água. No presente trabalho, detalha-se a montagem de um aparato proposto por TYREE et al. (2002), e sua calibração, bem como adaptações com peças de menor custo que tornam o aparelho acessível a qualquer um trabalhando nesta linha de pesquisa. Esse aparato possibilita medir a condutância de partes do sistema radicular ou da parte aérea (ramificações radiculares ou ramos), ou em todo o sistema, no caso de plantas de porte pequeno ou plântulas. O aparato também pode ser usado para medir a redução da condutância pela embolização dos vasos do xilema. Medições de condutância hidráulica feitas em plântulas de eucalipto e outros trabalhos encontrados na literatura confirmaram a aplicabilidade desse aparato em estudos fisiológicos de produtividade relacionada ao estresse hídrico.

Plant yield and resistance to drought are directly related to the efficiency of the xylem hydraulic conductance and the ability of this system to avoid interrupting the flow of water. In this paper we described in detail the assembling of an apparatus proposed by TYREE et al. (2002), and its calibration, as well as low cost adaptations that make the equipment accessible for everyone working in this research area. The apparatus allows measuring the conductance in parts of roots or shoots (root ramifications or branches), or in the whole system, in the case of small plants or seedlings. The apparatus can also be used to measure the reduction of conductance by embolism of the xylem vessels. Data on the hydraulic conductance of eucalyptus seedlings obtained here and other reports in the literature confirm the applicability of the apparatus in physiological studies on the relationship between productivity and water stress.

Algumas espécies de citros apresentam produção irregular ao longo dos anos. Além da safra irregular, a alternância de produção pode interferir na qualidade dos frutos, característica cada vez mais valorizada pelo mercado citrícola. O objetivo do trabalho foi avaliar o efeito da carga pendente na qualidade dos frutos da laranjeira 'Valência'. O experimento foi conduzido com laranjeiras 'Valência' enxertadas em tangerineiras 'Cleópatra' em condição de campo. Em janeiro de 2007, metade das plantas teve todos os seus frutos retirados, enquanto a outra metade permaneceu intacta. As plantas que tiveram seus frutos retirados apresentaram, em setembro de 2007, floração mais intensa e, portanto, uma Alta Carga de frutos (AC, 752 frutos/árvore) em 2008, quando comparadas às plantas que não tiveram seus frutos retirados, que apresentaram Baixa Carga de frutos (BC, 317 frutos/árvore). Os frutos maduros, colhidos em dezembro de 2008, foram analisados quanto à massa, teor de sólidos solúveis, acidez titulável, espessura e cor da casca, teor de suco e número de sementes. Na safra seguinte, de 2009, as avaliações das características físicoquímicas dos frutos foram repetidas nas mesmas árvores, mas durante quatro momentos entre o período de frutificação e a colheita, em novembro de 2009. Na safra de 2008, houve diferença na massa individual dos frutos com valores de 172 e 158g nas plantas BC e AC, respectivamente. O teor de sólidos solúveis, acidez titulável, teor de suco nos frutos, espessura da casca e o índice tecnológico foram semelhantes entre os tratamentos, nas safras de 2008 e 2009. Entretanto, vale considerar que, como a produção de frutos nas plantas AC, na safra de 2008, foi 2,2 vezes maior se comparada às das plantas BC (AC: 2,9 caixas/planta e BC: 1,3 caixa/planta), a quantidade total de sólidos solúveis produzida por planta também foi maior. Não houve influência da carga pendente de frutos no teor de açúcares (glicose, frutose e sacarose) no suco e na cor da casca durante o desenvolvimento dos frutos. Apesar de reduzir a massa do fruto, a diferença de carga pendente observada em laranjeira 'Valência' não influenciou na qualidade dos frutos quando considerado, como destino da fruta, a indústria citrícola.

Some citrus species show irregular production over the years. Besides the irregular crop yield, alternate bearing may affect the fruit quality, a characteristic that is increasingly valued by the citrus market. The aim of the work was to evaluate the effect of crop load on fruit quality of 'Valência' orange trees. The experiment was carried out under field conditions with 'Valência' orange scions grafted on 'Cleópatra' mandarin rootstocks. In January 2007, half of the plants were defruited, while the other half remained intact. Plants that had their fruit removed showed in September 2007 more intense flowering, and therefore a high fruit load (HL, 752 fruits/tree) in 2008, compared to intact plants showed low fruit load (LL, 317 fruits/tree). The fruits harvested in December 2008 were analyzed considering weight, soluble solids, titratable acidity, peel thickness, juice percentage, number of seeds and peel color. In the next crop, in 2009, the evaluations of physical and chemical characteristics of fruits were carried out in the same trees but during four moments between fruit development and harvest in November 2009. Fruit harvest in 2008 had different individual weight with values of 172 and 158 g for LL and HL plants, respectively. The soluble solids, titratable acidity, percentage of juice, peel thickness and technological index was similar between treatments in 2008 and 2009. However as fruit production in HL plants was 2.2 times higher than in LL plants in 2008 (AC: 2,9 boxes/plant and BC: 1,3 boxes/plant), total soluble solids produced per plant was also higher. Crop load did not affect the sugar content (glucose, fructose and sucrose) in juice and skin color during fruit development. Although it reduced the fruit weight, the difference in crop load observed in 'Valencia' orange had no influence on fruit quality when considered the citrus industry as fruit destination.

O chá preto é uma das bebidas mais consumidas no mundo. Essa bebida é feita a partir da infusão de folhas processadas de Camellia sinensis, que é cultivada em mais de 30 países, tendo grande importância socioeconômica. Estudos sugerem que o chá tem efeito protetor contra diversos tipos de câncer e doenças cardiovasculares devido à presença de polifenóis denominados catequinas, que são oxidadas enzimaticamente durante o processamento das folhas, gerando uma mistura constituída principalmente de teaflavinas, teasinensinas e tearubiginas. A produção mundial de chá tem aumentado mais do que o consumo, provocando redução do preço que, juntamente com o aumento no custo de produção, implica a necessidade de alta produtividade e qualidade. Apesar disso, ainda não foram estabelecidos critérios precisos e eficientes para predizer a qualidade do chá a partir das folhas, bem como quais práticas agronômicas contribuem para o aumento da qualidade. No Brasil, a cultura do chá se concentra no Vale do Ribeira, em São Paulo (SP), sendo quase toda produção exportada. Apesar de o produto brasileiro não ser de alta qualidade, tem conseguido bons preços no mercado internacional. A produção brasileira, a área de produção e o número de indústrias de chá vêm diminuindo ao longo dos últimos anos, o que demonstra a necessidade de investimentos.

Black tea is one of the most widely consumed beverages in the world and it is prepared by the infusion of water with processed leaves of Camellia sinensis, which is cultivated in more than 30 countries, assuming a great socioeconomic importance. Several studies suggest that tea has protective effects against cancer and cardiovascular disease, due to the presence of polyphenols called catechins, which are oxidized enzymatically during the processing of the black tea, generating a mixture consisting mainly of theaflavins, theasinensins and thearubigins. The world production of tea has increased more than the demand, causing a drop of the price which, together with the increase in cultivation costs has shown a need for high productivity and quality. Nevertheless, there is not yet an efficient and accurate criteria to predict the quality of tea and agronomic practices which contribute to the increase in quality. In Brazil, the culture of tea is concentrated in the Ribeira Valley - SP, and almost all the production is exported. Despite the Brazilian product is not of high quality, it has achieved good prices in the international market. The Brazilian production, the production area and the number of tea industries are decreasing in recent years, clearly indicating the need for investments.

Após a sua obtenção em laboratório, mudas in vitro necessitam passar por um período de aclimatização, pois na fase em que se encontram não realizam eficientemente a absorção de luz, água e nutrientes, devendo ser feita em ambiente protegido, onde as condições de temperatura, umidade e luminosidade são favoráveis a um gradual endurecimento das plântulas. No presente trabalho estudou-se o desenvolvimento das mudas de bananeira (Musa sp.) durante o segundo estágio da aclimatização (a partir de 10 cm de altura) sob condições de luminosidade (plena, 70, 50 e 30% de superfície de cobertura com malha vermelha e 50% com malha preta, ambas sob filme plástico transparente de 100 µm) associadas a períodos de aclimatização (três, seis e nove semanas), em ambientes com temperatura, umidade do ar, nutrição e irrigação controladas, sendo avaliadas sob variáveis físicas e bioquímicas. Imediatamente após cada período, as plantas foram submetidas a ensaios de campo por sete semanas, sendo avaliadas sob as mesmas variáveis. Na comparação entre as médias, cada tratamento recebeu pontuações obedecendo a ordenação hierárquica de desempenho, segundo critérios de classificação estatística onde se considera o número de tratamentos estatisticamente inferiores e superiores. Nesta ordenação, o uso de tela de malha preta com 50% de sombreamento associado ao período de nove semanas foi o que propiciou melhores condições, tanto no verão quanto no inverno. Visando a redução para seis semanas, o uso alternativo de tela de malha vermelha com 70% produz efeitos equivalentes ao da malha preta com 50%, apenas para condições de verão.

Banana plantlets obtained by micropropagation need to be submitted to a period of acclimatization since they do not use light, water, and nutrients in an efficient way. The acclimatization must be carried out under greenhouse conditions where temperature, light, and air humidity are adequate for a gradual hardening of the plantlets. In this study, the development of banana plantlets was evaluated during acclimatization under a full light condition including covered surfaces with red shade cloth (70%, 50%, and 30% shade) and black shade cloth (50% shade), both under a transparent plastic film of 100 µm. Temperature, relative air humidity, irrigation, and nutrition conditions were also controlled. Physical and physiological parameters were recorded at various stages in the greenhouses after three, six, and nine weeks and also after seven weeks of transplanting to field conditions. Treatments were hierarchically graded according to their statistic classification. Combined results indicated superior outcomes of plantlets maintained under black 50% shade cloth for nine weeks, both in the summer and winter seasons. Similar results, but in a shorter time, were obtained with plantlets cultivated under red 70% shade cloth, for six weeks in the summer.