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Cancer diseases have been widely recognized as a significant global public health problem and their incidence, morbidity and mortality are high in worldwide. There is a lot of evidence that fruit and vegetables could provide health benefits and reduce the risk of cancer. The anticancer properties of fruit and vegetables are attributed to their composition rich in phytochemicals or phytonutrients. This chapter will summarize the role of phytochemicals of fruit and vegetables namely quercetin, resveratrol, carotenoids and dietary fibers in cancer prevention. A large number of in vivo and in vitro experiments has demonstrated the beneficial effects of these compounds. These phytochemicals are also capable of increasing the effectiveness of drugs already established in treatments, reinforcing the importance of indicating the consumption of fruit and vegetables by the population.

Epidemiological studies have reported the importance of plant foods in distinct diets, because phytonutrients are related to health benefits and longevity. The intake of phytonutrients in plant food contributes to the reduction of risk factors for chronic diseases, such as cardiovascular diseases, diabetes, obesity, and cancer. To obtain a higher index of phytonutrients in the diet, it is recommended that one consume fruits, vegetables, and whole grains presenting their bioactive components with antiinflammatory, antitumoral, and antidiabetic properties, which could improve their beneficial action through synergistic effects, indicating the importance of whole plant food intake for maximum benefits to human health. However, the influence of environmental factors and the individual’s lifestyle factors, such as race, sex, age, and genetics, are still unclear. This chapter will highlight the effects of phytonutrients on the health status of populations.

The consumption of fruit has increased in the last 20 years, along with the growing recognition of its nutritional and protective values. Many of the benefits of a diet rich in fruit are attributed to the presence of different bioactive substances, such as vitamins, carotenoids and phenolic compounds. Flavanoids, a class of phenolic compounds, present particular antioxidant activity and thus provide protection against cellular damage caused by reactive oxygen species. Research suggests that an increased intake of plant foods is associated with a reduced incidence of chronic disease. There is currently a great deal of interest in the study of antioxidants, in particular due to the discovery of the damaging effects of free radicals to the body. Thus, this review aims to address the beneficial effects of the antioxidants present in fruits, on the neutralization of reactive species and the reduction of any damage they may cause.

Ginger is a plant whose rhizome or root is a rich source of phytochemical components such as zingiberene, α-curcumene, gingerols, and shogaols. These bioactive compounds are the major components in ginger dietary supplements and present biological and pharmacological antioxidant, antilipidemic, antihyperglycemic, antiinflammatory, antimicrobial, and anticancer activities. Although, ginger and its extracts are renowned for their contribution to human health, the pharmacokinetics of the biocompounds of ginger in human biological trials is not well studied. The coadministration of ginger and its products with other drugs could generate adverse effects in response to drug interaction, therefore, it should be recommended with care especially in the case of pregnant women. This chapter focuses on the main ginger phytochemical components as well as their medicinal properties and recommendations.

The consumption of the acai berry, from the acai palm tree (Euterpe oleracea) in the Amazon, as a functional food is widely used throughout the world. There is also a growing market for nutraceuticals and dietary supplements containing acai berries, which is claimed to have a large range of health-promoting and therapeutic benefits due to its extraordinary biological properties compared to other fruits. Studies published in the past on acai pulp or fruit berry extracts have revealed the biological effects associated with its phytochemical composition which is rich in polyphenols: anthocyanins and flavonoids. Cyanidin 3-glucoside, cyanidin 3-rutinoside, quercetin, orientin, and proanthocyanidins are the major polyphenols found in acai. Its biological effects include it being an antioxidant, an antiinflammatory, a cardioprotective, and a neuroprotective as well as improving dyslipidemia and obesity. Additional studies are required concerning its safe consumption as a dietary supplements.

Quantitative PCR (qPCR) is a robust technology for comparing the expression profiles of target genes, however the data produced need normalization with appropriate reference genes. The purpose of this study was to evaluate the expression stability of eight candidate reference genes (EF1A 1a1, EF1A 2a, EF1A 2b, EF1B, ACT11, UKN 1, ACT and SKIP 16) for qPCR assays in soybean leaves under fungus (Cercospora kikuchii) infection and treatments with salicylic and jasmonic acids. Four programs, GeNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the expression stability. The leaves were treated according the following conditions: 1) infected with Cercospora kikuchii (CK); (2) treated with salicylic acid (SA); (3) treated with SA and infected with C. kikuchii; (4) treated with jasmonic acid (JA); and (5) treated with JA and infected with C. kikuchii. For all studied conditions, GeNorm analyses revealed that combinations of six genes were always needed for gene expression normalization. Three EF1A genes (EF1A 2a, EF1A 1a1 and EF1A 2b) were the most stable in all tested conditions and then, they were always included in gene combinations. The other three genes varied according the different conditions. In analyses with other programs, at least two EF1A genes were often ranked among the three best stable genes. The expression of a PR3 (class I chitinase) gene was used to validate the reference genes across the total samples. Our results provide a shortlist of reference genes to normalize qPCR assays in soybean under CK infection and treatments with SA and JA.

International audience; The interactive effects of drought and ozone on the physiology and leaf membrane lipid content, composition and metabolism of cowpea (Vigna unguiculata (L.) Walp.) were investigated in two cultivars (EPACE-1 and IT83-D) grown under controlled conditions. The drought treatment (three-week water deprivation) did not cause leaf injury but restricted growth through stomatal closure. In contrast, the short-term ozone treatment (130 ppb 12 h daily during 14 day) had a limited impact at the whole-plant level but caused leaf injury, hydrogen peroxide accumulation and galactolipid degradation. These effects were stronger in the IT83-D cultivar, which also showed specific ozone responses such as a higher digalactosyl-diacylglycerol (DGDG):monogalactosyldiacylglycerol (MGDG) ratio and the coordinated up-regulation of DGDG synthase (VuDGD2) and ω-3 fatty acid desaturase 8 (VuFAD8) genes, suggesting that membrane remodeling occurred under ozone stress in the sensitive cultivar. When stresses were combined, ozone did not modify the stomatal response to drought and the observed effects on whole-plant physiology were essentially the same as when drought was applied alone. Conversely, the drought-induced stomatal closure appeared to alleviate ozone effects through the reduction of ozone uptake.

A classification scheme based on protein phylogenies and sequence harmony method was used to clarify the taxonomic distribution and evolutionary history of the alternative oxidase (AOX) in angiosperms. A large data set analyses showed that AOX1 and AOX2 subfamilies were distributed into 4 phylogenetic clades: AOX1a-c/1e, AOX1d, AOX2a-c and AOX2d. High diversity in AOX family compositions was found. While the AOX2 subfamily was not detected in monocots, the AOX1 subfamily has expanded (AOX1a-e) in the large majority of these plants. In addition, Poales AOX1b and 1d were orthologous to eudicots AOX1d and then renamed as AOX1d1 and 1d2. AOX1 or AOX2 losses were detected in some eudicot plants. Several AOX2 duplications (AOX2a-c) were identified in eudicot species, mainly in the asterids. The AOX2b originally identified in eudicots in the Fabales order (soybean, cowpea) was divergent from AOX2a-c showing some specific amino acids with AOX1d and then it was renamed as AOX2d. AOX1d and AOX2d seem to be stress-responsive, facultative and mutually exclusive among species suggesting a complementary role with an AOX1(a) in stress conditions. Based on the data collected, we present a model for the evolutionary history of AOX in angiosperms and highlight specific areas where further research would be most beneficial.

Lead (Pb) is one of the most toxic anthropogenic pollutants, occurring widely in both terrestrial and aquatic ecosystems, where it impairs plant growth and development. In this work, the effect of 0.5 mM EDTA-Pb was evaluated in two Vigna unguiculata cultivars (SV and SET), with the aim of detecting genotype/cultivar dependent changes in the physiological and anti-oxidant responses (CAT and APX) of a leguminous plant. The data showed that SV accumulated more Pb in roots while SET accumulated more in leaves, indicating differential regulation in Pb-translocation/accumulation. Lead affected the growth of SV less severely than SET, mainly associated with reduced inhibition in photosynthetic parameters. Furthermore, CAT and APX activities increased or were sustained at elevated levels in both cultivars in response to lead. However, gene expression analyses revealed that CAT1 was the main lead responsive gene in SET while CAT2 was more responsive in SV. APX1 was higher expressed in tissues with higher Pb-accumulation while APX2 was ubiquitously responsive to lead in both cultivars. Taken together, these results reveal differential ability of V. unguiculata cultivars in Pb-accumulation in different tissues affecting distinctly physiological and anti-oxidant responses. In addition, the existence of cultivars with predominant Pb-accumulation in aerial tissues invokes a need for studies to identify pollution-safe cultivars of leguminous plants to ensure food safety.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:43:58Z No. of bitstreams: 1 S1806-66902011000200020.pdf: 299400 bytes, checksum: 89aac4b1cee0e68680f62a7f25e0b974 (MD5) Made available in DSpace on 2013-08-22T18:43:58Z (GMT). No. of bitstreams: 1 S1806-66902011000200020.pdf: 299400 bytes, checksum: 89aac4b1cee0e68680f62a7f25e0b974 (MD5) Previous issue date: 2011-06-01 Made available in DSpace on 2013-09-30T19:31:55Z (GMT). No. of bitstreams: 2 S1806-66902011000200020.pdf: 299400 bytes, checksum: 89aac4b1cee0e68680f62a7f25e0b974 (MD5) S1806-66902011000200020.pdf.txt: 23797 bytes, checksum: 67b14c7a6ef2b5ea95b07b03d34405a7 (MD5) Previous issue date: 2011-06-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:50:19Z No. of bitstreams: 2 S1806-66902011000200020.pdf: 299400 bytes, checksum: 89aac4b1cee0e68680f62a7f25e0b974 (MD5) S1806-66902011000200020.pdf.txt: 23797 bytes, checksum: 67b14c7a6ef2b5ea95b07b03d34405a7 (MD5) Made available in DSpace on 2014-05-20T13:50:19Z (GMT). No. of bitstreams: 2 S1806-66902011000200020.pdf: 299400 bytes, checksum: 89aac4b1cee0e68680f62a7f25e0b974 (MD5) S1806-66902011000200020.pdf.txt: 23797 bytes, checksum: 67b14c7a6ef2b5ea95b07b03d34405a7 (MD5) Previous issue date: 2011-06-01 Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP) NTCSal Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) O estresse salino afeta o crescimento e o desenvolvimento das plantas, induzindo respostas bioquímicas e fisiológicas como mecanismo de adaptação para sua sobrevivência. As proteínas desacopladoras de planta (pUCPs) dissipam o gradiente eletroquímico mitocondrial de prótons como calor e são codificadas por famílias multigênicas. Elas atuam como sistema de defesa evitando a formação de espécies reativas de oxigênio geradas em resposta a estresses ambientais. O objetivo do trabalho foi estudar a expressão de genes das pUCPs (VuUCP1a e VuUCP1b ) em raízes e folhas de plântulas de Vigna unguiculata submetidas ao estresse salino (NaCl 100 mM). Sementes foram germinadas no escuro e após 3 dias, as plântulas foram transferidas para solução de Hoagland, permanecendo 3 dias antes da aplicação do estresse. As raízes e folhas foram coletadas com 0; 6; 12 e 24 horas, após adição de NaCl, para avaliar o perfil de expressão dos genes da pUCP por RT- PCR. A análise de transcritos mostrou que VuUCP1a é expresso em raízes e folhas revelando um perfil constitutivo enquanto que VuUCP1b é dependente do tecido, isto é, expresso nas folhas, sem alteração em resposta ao estresse e induzido pelo estresse salino nas raízes. A peculiaridade da duplicação gênica de pUCP1 em caupi com perfil diferencial de expressão, sugere tanto um papel dessa enzima nos mecanismos de ajustamento ao estresse salino quanto promove essa espécie como um modelo atrativo para compreensão do papel da família multigênica da pUCP em plantas. Salt stress affects growth and development of plants, inducing a variety of physiological and biochemical responses as an adaptation mechanism for survival. The plant uncoupling mitochondrial proteins (pUCPs) are able to dissipate the proton electrochemical gradient as heat and are encoded by a multigene family. They works as defence systems avoiding the formation of reactive oxygen species promoted by environmental stress. The aim of this work was to study gene expression of pUCPs (VuUCP1a and VuUCP1b) in roots and leaves from Vigna unguiculata seedlings under salt stress (100 mM NaCl). Seeds were germinated in the dark and after 3 days, the seedlings were transferred to Hoagland's medium and grown for 3 additional days before being submitted to the stress condition. Roots and leaves were harvested at 0; 6; 12 and 24 hours after addition of NaCl for total RNA isolation and RT-PCR assays. Expression analysis by RT-PCR showed that VuUCP1a is constitutive in leaves and roots while VuUCP1b is expressed as tissue-dependent presenting a constitutive profile in leaves and a differential one in roots from seedlings under salt stress. The uniqueness of pUCP1 gene duplication in cowpea with differential expression suggest a role of this enzyme in the adjustment of salt stress as well as promotes this species as an attractive model to understand the role of pUCP gene members in plants. UFC Centro de Ciências Departamento de Bioquímica e Biologia Molecular Universidade Estadual Paulista Instituto de Biociências Departamento de Genética Universidade Estadual Paulista Instituto de Biociências Departamento de Genética

Vigna unguiculata (cowpea) is a legume adapted to high temperatures and is sensitive to low temperatures. Temperature is one of the limiting factors of growth and yield for many crops but its effect on cowpea metabolism is not known. We investigated the effect of chilling on activity of vacuolar proton pumps (V-ATPase and V-PPase) and their protein content in tonoplast vesicles of cowpea hypocotyls. Seedlings grown for 7 days at 10 or 4°C were used for experiments. Chilling treatment at 10 or 4°C markedly suppressed growth of cowpea seedlings. Following chilling at 10 and 4°C, activity of both proton pumps and the relative amount of V-PPase and subunit A of V-ATPase were significantly increased. Both substrate hydrolysis and H+ transport activities of V-PPase remained at relatively high levels during chilling treatment. For V-ATPase, treatment at 10°C for 6 days increased the ATP hydrolysis activity. However, the H+ transport activity of the enzyme was increased when treated for 4 days but was markedly decreased when treated for 6 days. Our results provide evidence for different regulation for these vacuolar proton pumps, indicating that V-PPase is the more stable proton pump throughout chilling stress.

The present study was undertaken to investigate the changes induced by chilling on fatty acid composition of tonoplast vesicles from hypocotyls of Vigna unguiculata (L.) Walp. The 7 day-old control seedlings were grown at 25°C while treated seedlings were submitted to low temperatures (10°C and 4°C) for 4 days after 3 days germination. The chilling stress resulted in a differential inhibition of plant growth at 10°C and 4°C. Following chilling at 10°C and 4°C, the rate of unsaturated to saturated fatty acids increased under chilling stress. Our results suggest that the increase of unsaturated fatty acids content as well as the higher rate unsaturated/saturate fatty acidsmight be used by V. unguiculata plants as an adaptation mechanism likely to maintain the vacuolar membrane fluidity under low temperatures.

Aims: The purpose of this study was to demonstrate the usefulness of lectin obtained from Talisia esculenta (TEL) seeds as a tool to recognize and study Microsporum canis. For this purpose, we investigated the antifungal and marker action of this lectin and the relationship of these effects with the presence of carbohydrates on the structure of this fungus. Methods and Results: The in vitro antifungal activity of TEL was analysed by broth microdilution assay. In addition, TEL was assessed against the arthroconidia present on hairs obtained from infected dogs and cats. The affinity of fluorescein isothiocyanate (FITC)-labelled TEL for macroconidia and arthroconidia of M. canis was also tested. The effects of TEL on the growth of the M. canis strains began with 0·125 mg ml−1, and 100% inhibition was obtained with a concentration of 2 mg ml−1. The addition of carbohydrates, especially N-acetyl-glucosamine and d-mannose, inhibited these antifungal effects. TEL was able to inhibit the growth of arthroconidial chitin-rich forms of M. canis obtained from hairs of infected animals and strains cultured in Sabouraud agar. FITC-labelled TEL efficiently marked macroconidial and arthroconidial forms of M. canis, as shown by fluorescent microscopy. Conclusions: These results show that the inhibitory effects of TEL on M. canis growth may be related to the interaction of lectin with the carbohydrates present at the micro-organism’s surface, mainly d-mannose and N-acetyl-glucosamine. Significance and Impact of the Study: Talisia esculenta can be used as an important tool in the biochemical study of M. canis or as a molecule to recognize this dermatophyte in infected tissue.

Alginate fractions from Sargassum vulgare brown seaweed were characterized by (1)H NMR and fluorescence spectroscopy and by rheological measurements. The alginate extraction conditions were investigated. In order to carry out the structural and physicochemical characterization, samples extracted for 1 and 5h at 60 degrees C were further purified by re-precipitation with ethanol and denoted as SVLV (S. vulgare low viscosity) and SVHV (S. vulgare high viscosity), respectively. The M/G ratio values for SVLV and SVHV were 1.56 and 1.27, respectively, higher than the ratio for most Sargassum spp. alginates (0.19-0.82). The homopolymeric blocks F(GG) and F(MM) of these fractions characterized by (1)H NMR spectroscopy were 0.43 and 0.55 for SVHV and 0.36 and 0.58 for SVLV samples, respectively, these values typically being within 0.28-0.77 and 0.07-0.41, respectively. Therefore, the alginate samples from S. vulgare are much richer in mannuronic block structures than those from other Sargassum species. Values of M(w) for alginate samples were also calculated using intrinsic viscosity data. The M(w) value for SVLV (1.94 x 10(5)g/mol) was lower than that for SVHV (3.3 x 10(5)g/mol). Newtonian behavior was observed for a solution concentration as high as 0.7% for SVLV, while for SVHV the solutions behaved as a Newtonian fluid up to 0.5%. The optimal conditions for obtaining the alginates from S. vulgare were 60 degrees C and 5h extraction. Under these conditions, a more viscous alginate in higher yield was extracted from the seaweed biomass.

Enhanced respiration during ripening in climacteric fruits is sometimes associated with an uncoupling between the ATP synthesis and the mitochondrial electron transport chain. While the participation of two energy-dissipating systems, one of which is mediated by the alternative oxidase (AOX) and the other mediated by the uncoupling protein (UCP), has been linked to fruit ripening, the relation between the activation of both mitochondrial uncoupling systems with the transient increase of ethylene synthesis (ethylene peak) remains unclear. To elucidate this question, ethylene emission and the two uncoupling (AOX and UCP) pathways were monitored in harvested papaya fruit during the ripening, from green to fully yellow skin. The results confirmed the typical climacteric behavior for papaya fruit: an initial increase in endogenous ethylene emission which reaches a maximum (peak) in the intermediate ripening stage, before finally declining to a basal level in ripe fruit. Respiration of intact fruit also increased and achieved higher levels at the end of ripening. On the other hand, in purified mitochondria extracted from fruit pulp the total respiration and respiratory control decrease while an increase in the participation of AOX and UCP pathways was markedly evident during papaya ripening. There was an increase in the AOX capacity during the transition from green fruit to the intermediate stage that accompanied the transient ethylene peak, while the O2 consumption triggered by UCP activation increased by 80% from the beginning to end stage of fruit ripening. Expression analyses of AOX (AOX1 and 2) and UCP (UCP1-5) genes revealed that the increases in the AOX and UCP capacities were linked to a higher expression of AOX1 and UCP (mainly UCP1) genes, respectively. In silico promoter analyses of both genes showed the presence of ethylene-responsive cis-elements in UCP1 and UCP2 genes. Overall, the data suggest a differential activation of AOX and UCP pathways in regulation related to the ethylene peak and induction of specific genes such as AOX1 and UCP1.

The EF1α is a multifunctional protein with additional unrelated activities to its primary function in translation. This protein is encoded by a multigene family and few studies are still available in plants. Expression of six EF1α genes in Glycine max was performed using RT-qPCR and RNA-seq data to advance in the function of each gene during plant development, stress conditions and phytohormone treatments. A phylogenetic classification in Phaseoleae tribe was used to identify the G. max EF1α genes (EF1α 1a1, 1a2, 1b, 2a, 2b and 3). Three EF1α types (1-3) were found in Phaseoleae revealing duplications in G. max types 1 and 2. EF1α genes were expressed in all studied tissues, however, specific amount of each transcript was detected. In plant development, all EF1α transcripts were generally more expressed in younger tissues, however, in unifoliolate leaves and cotyledons a higher expression occurred in older tissues. Five EF1α genes (except 2a) were up-regulated under stress in a response tissue/stress/cultivar-dependent. EF1α 3 was the most stress-induced gene linked to cultivar stress tolerance mainly in aerial tissues. Auxin, salicylate and ethylene induced differentially the EF1α expression. Overall, this study provides a consistent EF1α classification in Phaseoleae tribe to better understand their functional evolution. The RT-qPCR and RNA-seq EF1α expression profiles were consistent, both exhibiting expression diversification of each gene (spatio-temporal, stress and phytohormone stimuli). Our results point out the EF1α genes, especially EF1α 3, as candidate for developing a useful tool for future G. max breeding.

Cashew apple is a tropical pseudofruit consumed as juice due to its excellent nutritional and sensory properties. In spite of being well known for its important antioxidant properties, the cashew apple has not been thoroughly investigated for its therapeutic potential. Thereby, this study evaluated the antioxidant capacity, anti-inflammatory, and wound-healing activities of cashew apple juice. Juices from ripe and immature cashew apples were analyzed for antioxidant, anti-inflammatory, and wound-healing properties. Those were evaluated in murine models of xylene-induced ear edema and wound excision. Swiss mice were treated with cashew juice by gavage. Edema thickness was measured and skin lesions were analyzed by planimetry and histology. Both antioxidant content and total antioxidant activity were higher in ripe cashew apple juice (RCAJ) than in unripe cashew apple juice (UNCAJ). The UNCAJ presented the main anti-inflammatory activity by a significant inhibition of ear edema (66.5%) when compared to RCAJ (10%). Moreover, UNCAJ also showed the best result for wound contraction (86.31%) compared to RCAJ (67.54%). Despite of higher antioxidant capacity, RCAJ did not promote better anti-inflammatory, and healing responses, which may be explained by the fact that treatment increased antioxidants level leading to a redox “imbalance” turning down the inflammatory response modulation exerted by reactive oxygen species (ROS). The results suggest that UNCAJ presents a greater therapeutic activity due to a synergistic effect of its phytochemical components, which improve the immunological mechanisms as well as an optimal balance between ROS and antioxidants leading to a better wound healing process.

Global climatic changes as high temperatures and low precipitation contribute to increase cultivated areas affected by high salt soil content. Soil salinity is well known to reduce the ability of plants to take up water and this quickly causes reduction in their growth rate. V-ATPase (EC 3.6.3.14) and V-PPase (EC 3.6.1.1) hydrolytic and proton transport activities, and gene expression were evaluated in hypocotyls of 3-, 5-, 7-day-old Vigna unguiculata (L.) Walp cv. Vita 3 germinated in 100 mM NaCl in order to highlight their differential regulation and activity modulation under salt stress. Semi-quantitative RT-PCR revealed that both genes were up-regulated by salt stress in all salt exposition times studied. Up-regulation was correlated with the increase in protein content at 5 and 7-day-old seedlings. Co-expression between A and E V-ATPase subunits was also observed. The hydrolytic and proton transport activities showed that these enzymes presented a differential modulation of their activities in the presence of 100 mM NaCl. These results suggest that V-ATPase and V-PPase activities are modulated by salt stress and a multi-step regulation is exerted in order to re-establish homeostasis Fil: Menezes Sobreira, Alana Cecília. Universidade Estadual Do Ceara; Brasil. Universidade Federal Do Ceara; Brasil Fil: Maia, Yuri. Universidade Federal Do Ceara; Brasil Fil: Rebouças, Deborah Moura. Universidade Federal Do Ceara; Brasil Fil: Costa, José Hélio. Universidade Federal Do Ceara; Brasil Fil: Oliveira Otoch, Maria de Lourdes. Universidade Estadual Do Ceara; Brasil Fil: Nogueira de Oliveira, Luciana Maia. Universidade Federal Rural Pernambuco; Brasil Fil: Orellano, Elena Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Fernandes de Melo, Dirce. Universidade Federal do Ceará; Brasil

Summary Salt modulation of the tonoplast H+-pumping V-ATPase and H+-PPase was evaluated in hypocotyls ofVigna unguiculata seedlings after 3 and 7 days of treatment. In 3-day-old seedlings, treatment with 100 mmol/L NaCl decreased the proton transport and hydrolytic activities of both the V-ATPase and the H+-PPase. After 7 days, the proton transport and hydrolysis activities of the V-ATPase were higher, while the H+-PPase activities were lower in seedlings. Western blot analysis of A- and B-subunits of V-ATPase revealed that the protein content of the two subunits varied in parallel with their activities, i.e. to a higher activity corresponded a higher protein content of the subunits and vice versa. Contrarily, Western blot analysis of H+-PPase levels failed to show any correlation with PPase activity, suggesting a partial enzyme inactivation. The results indicate that salt stress induces V-ATPase expression inV. unguiculata with concomitant enhancement of its activity as a homeostatic mechanism to cope with salt stress. Under the same conditions PPase is inhibited.

Mitochondria from Vigna unguiculata cv. Vita 5 have a cyanide insensitive alternative oxidase cross-reacting with a monoclonal antibody raised against the alternative oxidase of Sauromatum guttatum. In the presence of NADH as substrate and dithiothreitol, the CN-resistant respiration of V. unguiculata intact mitochondria was found to be significantly influenced by assay pH with an optimum value of 6.25. This effect was still observed when nigericin, known to abolish ΔpH between the matrix and the intermembrane space, was present in the reaction medium. This pH effect was shown to be reversible. Alternative oxidase activation by pyruvate, also appeared to be pH dependent with an optimum pH value of 7.25. This modulation of the alternative oxidase activity by pH has been tentatively attributed to a new protonated-deprotonated status of this protein.

The EF1α genes were stable in the large majority of soybean tissues during development and in specific tissues/conditions under stress. Quantitative real-time PCR (qPCR) analysis strongly depends on transcript normalization using stable reference genes. Reference genes are generally encoded by multigene families and are used in qPCR normalization; however, little effort has been made to verify the stability of different gene members within a family. Here, the expression stability of members of the soybean EF1α gene family (named EF1α 1a1, 1a2, 1b, 2a, 2b and 3) was evaluated in different tissues during plant development and stress exposure (SA and PEG). Four genes (UKN1, SKIP 16, EF1β and MTP) already established as stably expressed were also used in the comparative analysis. GeNorm analyses revealed different combinations of reference genes as stable in soybean tissues during development. The EF1α genes were the most stable in cotyledons (EF1α 3 and EF1α 1b), epicotyls (EF1α 1a2, EF1α 2b and EF1α 1a1), hypocotyls (EF1α 1a1 and EF1β), pods (EF1α 2a and EF1α 2b) and roots (EF1α 2a and UKN1) and less stable in tissues such as trifoliate and unifoliate leaves and germinating seeds. Under stress conditions, no suitable combination including only EF1α genes was found; however, some genes were relatively stable in leaves (EF1α 1a2) and roots (EF1α 1a1) treated with SA as well as in roots treated with PEG (EF1α 2b). EF1α 2a was the most stably expressed EF1α gene in all soybean tissues under stress. Taken together, our data provide guidelines for the selection of EF1α genes for use as reference genes in qPCR expression analyses during plant development and under stress conditions.

Three-month-old plants of Eucalyptus citriodora were grown in a greenhouse in a culture medium adjusted either to 50 or 100 mM NaCl. Sodium was rapidly absorbed by the plants and as early as 3 weeks after treatment, an increased Na+ level was observed in shoots of 50 mM NaCl plants. At this stage of treatment, the growth of plants was not reduced but the malate metabolism was modified. The malate content decreased in leaves while the specific activities of NAD and NADP-malic enzymes increased. The stimulation in enzyme activity was more pronounced for NADP-malic enzyme but for both enzymes, enzyme activity diminished as early as 5 weeks after treatment. The immunological study showed that the higher activity of NAD-malic enzyme was linked to an increase in the protein amount. By contrast, a more active form of the NADP-malic enzyme mainly accounted for the higher activity of this enzyme without involving modification in the electrophoretic mobility of the protein. The 100 mM NaCl treatment also induced changes in the malic enzyme behaviour, the magnitude of the response however being less than for the 50 mM treatment. These results are discussed, mainly considering the role of these enzymes in the reducing power of the leaf cell.

Aim and Background Acerola (Malpighia emarginata DC.) is a tropical fruit known for its nutritional and functional properties due to its great contents vitamin C, carotenoids and anthocyanins. The antioxidant potential from plants extracts associated to hepatoprotective activity has been widely studied. However, the effect of antioxidants in natural fruit juice has not been fully searched. The aim of this work was to investigate the antioxidant properties of acerola juice and its hepatoprotective potential against acute ethanol-induced stress. Materials and methods Ripe acerola were collected, processed into juice and initially analyzed to antioxidant properties in vitro. Afterwards, in vivo hepatoprotective activity was evaluated in mice. The animals received the juice by gavage as pretreatment for 15 consecutive days and then, were submitted to ethanol-induced stress in single dose (5 g/kg). The activities of serum enzymes as well as lipid peroxidation degree were evaluated. The activities of serum marker enzymes for liver damage as well as lipid peroxidation degree were evaluated. Results Acerola juice presents great vitamin C (1799.5 mg/100 g FW) and total phenolic (188.4 mg GAE/100 g FW), anthocyanins (9.2 mg/100 g FW), flavonols (7.8 mg/100 g FW) contents and high activity of superoxide dismutase (1053.6 UA/g DM) with a total antioxidant activity of 137.5 μmol Trolox/g FW. The juice treatment inhibited lipid peroxidation and reduced the activities of aminotransferases (p ≤0.05), in mice liver. Conclusion These results indicate that acerola juice is able to prevent the hepatic damage induced by ethanol, probably as a result of an enhancement of the antioxidant status in the animals.

In flowering plants, alternative oxidase (Aox) is encoded by 3-5 genes distributed in 2 subfamilies (Aox1 and Aox2). In several species only Aox1 is reported as a stress-responsive gene, but in the leguminous Vigna unguiculata Aox2b is also induced by stress. In this work we investigated the Aox genes from two leguminous species of the Medicago genus (Medicago sativa and Medicago truncatula) which present one Aox1, one Aox2a and an Aox2b duplication (named here Aox2b1 and Aox2b2). Expression analyses by semi-quantitative RT-PCR in M. sativa revealed that Aox1, Aox2b1 and Aox2b2 transcripts increased during seed germination. Similar analyses in leaves and roots under different treatments (SA, PEG, H2O2 and cysteine) revealed that these genes are also induced by stress, but with peculiar spatio-temporal differences. Aox1 and Aox2b1 showed basal levels of expression under control conditions and were induced by stress in leaves and roots. Aox2b2 presented a dual behavior, i.e., it was expressed only under stress conditions in leaves, and showed basal expression levels in roots that were induced by stress. Moreover, Aox2a was expressed at higher levels in leaves and during seed germination than in roots and appeared to be not responsive to stress. The Aox expression profiles obtained from a M. truncatula microarray dataset also revealed a stress-induced co-expression of Aox1, Aox2b1 and Aox2b2 in leaves and roots. These results reinforce the stress-inducible co-expression of Aox1/Aox2b in some leguminous plants. Comparative genomic analysis indicates that this regulation is linked to Aox1/Aox2b proximity in the genome as a result of the gene rearrangement that occurred in some leguminous plants during evolution. The differential expression of Aox2b1/2b2 suggests that a second gene has been originated by recent gene duplication with neofunctionalization.

Alternative oxidase (AOX) is a mitochondrial protein encoded by the nuclear genome. In higher plants AOX genes form a small multigene family mostly consisting of the two subfamilies AOX1 and AOX2. Daucus carota L. is characterized by a unique extension pattern of AOX genes. Different from other plant species studied so far it contains two genes in both subfamilies. Therefore, carrot was recently highlighted as an important model in AOX stress research to understand the evolutionary importance of both AOX subfamilies. Here we report on the expression patterns of DcAOX1a, DcAOX1b and DcAOX2a and DcAOX2b. Our results demonstrate that all of the four carrot AOX genes are expressed. Differential expression was observed in organs, tissues and during de novo induction of secondary root phloem explants to growth and development. DcAOX1a and DcAOX2a indicated a differential transcript accumulation but a similar co-expression pattern. The genes of each carrot AOX sub-family revealed a differential regulation and responsiveness. DcAOX2a indicated high inducibility in contrast to DcAOX2b, which generally revealed low transcript abundance and rather weak responses. In search for within-gene sequence differences between both genes as a potential reason for the differential expression patterns, the structural organization of the two genes was compared. DcAOX2a and DcAOX2b showed high sequence similarity in their open reading frames (ORFs). However, length variability was observed in the N-terminal exon1 region. The predicted cleavage site of the mitochondrial targeting sequence in this locus is untypical small for both genes and consists of 35 amino acids for DcAOX2a and of 21 amino acids for DcAOX2b. The importance of structural gene organization and the relevancy of within-gene sequence variations are discussed. Our results strengthen the value of carrot as a model plant for future studies on the importance of AOX sub family evolution.

'Genomic design' refers to the structural organization of gene sequences. Recently, the role of intron sequences for gene regulation is being better understood. Further, introns possess high rates of polymorphism that are considered as the major source for speciation. In molecular breeding, the length of gene-specific introns is recognized as a tool to discriminate genotypes with diverse traits of agronomic interest. 'Economy selection' and 'time-economy selection' have been proposed as models for explaining why highly expressed genes typically contain small introns. However, in contrast to these theories, plant-specific selection reveals that highly expressed genes contain introns that are large. In the presented research, 'wet'Aox gene identification from grapevine is advanced by a bioinformatics approach to study the species-specific organization of Aox gene structures in relation to available expressed sequence tag (EST) data. Two Aox1 and one Aox2 gene sequences have been identified in Vitis vinifera using grapevine cultivars from Portugal and Germany. Searching the complete genome sequence data of two grapevine cultivars confirmed that V. vinifera alternative oxidase (Aox) is encoded by a small multigene family composed of Aox1a, Aox1b and Aox2. An analysis of EST distribution revealed high expression of the VvAox2 gene. A relationship between the atypical long primary transcript of VvAox2 (in comparison to other plant Aox genes) and its expression level is suggested. V. vinifera Aox genes contain four exons interrupted by three introns except for Aox1a which contains an additional intron in the 3'-UTR. The lengths of primary Aox transcripts were estimated for each gene in two V. vinifera varieties: PN40024 and Pinot Noir. In both varieties, Aox1a and Aox1b contained small introns that corresponded to primary transcript lengths ranging from 1501 to 1810 bp. The Aox2 of PN40024 (12 329 bp) was longer than that from Pinot Noir (7279 bp) because of selection against a transposable-element insertion that is 5028 bp in size. An EST database basic local alignment search tool (BLAST) search of GenBank revealed the following ESTs percentages for each gene: Aox1a (26.2%), Aox1b (11.9%) and Aox2 (61.9%). Aox1a was expressed in fruits and roots, Aox1b expression was confined to flowers and Aox2 was ubiquitously expressed. These data for V. vinifera show that atypically long Aox intron lengths are related to high levels of gene expression. Furthermore, it is shown for the first time that two grapevine cultivars can be distinguished by Aox intron length polymorphism.

Membranes are main targets of drought, and there is growing evidence for the involvement of membrane lipid in plant adaptation to such an environmental stress. Biosynthesis of the galactosylglycerolipids, monogalactosyl-diacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG), which are the main components of chloroplast envelope and thylakoid membranes, could be important for plant tolerance to water deficit and for recovery after rehydration. In this study, galactolipid (GL) biosynthesis in cowpea (Vigna unguiculata L. Walp) leaves was analysed during drought stress and subsequent rewatering. Comparison of two cowpea cutivars, one drought tolerant and the other drought susceptible submitted to moderate drought stress, revealed patterns associated with water-deficit tolerance: increase in DGDG leaf content, stimulation of DGDG biosynthesis in terms of (14)C-acetate incorporation and messenger accumulation corresponding to four genes coding for GL synthases (MDG1, MGD2, DGD1 and DGD2). Similar to phosphate starvation, lack of water enhanced DGDG biosynthesis and it was hypothesized that the drought-induced DGDG accumulated in extrachloroplastic membranes, and thus contributes to plant tolerance to arid environments.

International audience; The alternative oxidase (Aox) was studied at different levels (transcript, protein and capacity) in response to an osmotic shock applied to roots of cowpea (Vigna unguiculata). Two cultivars of V. unguiculata were used, Vita 3 and Vita 5, tolerant and sensitive to drought/saline stress respectively. The seedlings (17-day-old) were grown in hydroponic conditions and submitted to NaCl (100 and 200mM) or 200.67gL-1 PEG 6000 (iso-osmotic condition to 100mMNaCl). The VuAox1 and VuAox2a mRNA were not detected in either cultivar under all tested conditions while the VuAox2b gene was differently expressed. In the tolerant cultivar (Vita 3), the expression of VuAox2b gene was stimulated by an osmotic stress induced by PEG which was associated with a higher amount and capacity of the Aox protein. In the same cultivar, this gene was under-expressed in salt stress conditions with poor effect on the protein level. In the sensitive cultivar (Vita 5), the transcript level of the VuAox2b was unchanged in response to PEG treatment, even though the protein and the capacity tended to increase. Upon salt stress, the VuAox2b gene was over-expressed. At 100mM NaCl, this VuAox2b gene over-expression led to a higher amount and capacity of Aox. This effect was reduced at 200 mM NaCl. Overall, these results suggest complex mechanisms (transcriptional, translational and post-translational) for Aox regulation in response to osmotic stress.

Functional markers for stress tolerance can be used in plant breeding to identify genotypes with high yield stabilities under various conditions. Thus, a good marker should show a strong correlation with favourable adaptive plant behaviour. The efficient reprogramming of target cells for yield determination is currently considered to be the most important step towards defining abiotic stress tolerance. In this Opinion article, we propose a role for the alternative oxidase (AOX) gene as a marker for genetic variation in cell reprogramming and yield stability. Evidence to support this idea comes from the metabolic role of alternative respiration under stress, the link between AOX activity and differential growth, and the single nucleotide polymorphism recently observed in AOX genes. We propose an innovative, interdisciplinary and global research strategy for future experimentation on AOX genes that could have an application in plant breeding.

Vita 3 and Vita 5 are two Vigna unguiculata cultivars that differ in their capacities for survival in saline environments; Vita 3 is more tolerant and Vita 5 more sensitive. Both cultivars were submitted to salt stress with 0.1 M NaCl. After 8 days, root and shoot growth from both cultivars was reduced but reduction was more pronounced in Vita 5. Furthermore, leaf area was also reduced in this cultivar. Chlorophyll content and chlorophyll fluorescence parameters were not affected by salt stress, but the specific activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) decreased in Vita 3 and increased in Vita 5. The use of immunological techniques also revealed that the Rubisco content from Vita 3 decreased while that of Vita 5 increased. The discussion of these results is aimed at reaching a better understanding of the differences between these cultivars in relation to salt stress. Vita 3 e Vita 5 são cultivares de Vigna unguiculata que diferem na capacidade de sobrevivência em ambiente salino: Vita 3 é mais tolerante e, Vita 5, mais suscetível. Ambas as cultivares foram submetidas a um tratamento salino com NaCl 0,1 M. Após 8 dias da germinação, o crescimento das raízes e das partes aéreas foram reduzidas, sendo a redução mais acentuada na cultivar Vita 5. Além do mais, houve uma diminuição na sua área foliar. Os conteúdos de clorofila, bem como seus parâmetros de fluorescência não foram afetados pelo estresse salino, mas as atividades da ribulose-1,5-bisfosfato carboxilase/oxigenase (Rubisco) diminuiu na Vita 3 e aumentou na Vita 5. Uma abordagem imunológica mostrou que o conteúdo da proteína Rubisco diminuiu para a cultivar Vita 3 e aumentou para a cultivar Vita 5. Esses resultados são discutidos para obter melhor entendimento das diferenças entre essas cultivares em relação ao estresse salino.

Plant mitochondria contain an alternative oxidase (Aox) that catalyses cyanide-resistant oxygen consumption. Two clones, encoding putative Aox proteins (VuAox2a and VuAox2b), were isolated from a Vigna unguiculata cDNA library and sequenced. Based on deduced amino acid sequences, we have shown that VuAox2a and VuAox2b are orthologous to soybean GmAox2a (90%) and GmAox2b (91%), respectively. RT-PCR analyses revealed that Aox2b was expressed in all tissues from seedlings developed in etiolated or light conditions, with a higher level of expression in hypocotyls. In addition, Aox2a, although mainly expressed in leaves, was also found in hypocotyls at a low level of expression in light conditions. Thus, the pattern of expression of the two V. unguiculata Aox genes, described here for the first time, is shown to be similar to that of the orthologous genes found in soybean.

Mitochondria from the bean Vigna unguiculata contain an ATPase system that is strongly activated by K+ and Na+ in the presence of low concentrations of substrate. Optimum activation by physiological concentrations of K+ is achieved in the presence of 0.1 mM MgATP. At the substrate optimum of the total ATPase, which is 2.3 mM, the enzyme is only poorly activated by K+. The K+-activated component of the total F1-ATPase behaves like an individual enzyme with kinetic properties different to the total F1-ATPase. On the basis of enzyme kinetic measurements, we determined the mechanism of activation by K+ as follows:[Formula: see text]K+ is not required for the binding of the substrate MgATP to the enzyme but for the splitting of the enzyme–substrate complex into the reaction products.

Beef heart mitochondrial F1-ATPase was inactivated by the 2′,3′-dialdehyde derivatives of ATP, ADP and AMP (oATP, oADP, oAMP). In the absence of Mg2+, inactivation resulted from the binding of 1 mol nucleotide analog per active unit of F1. The most efficient analog was oADP, followed by oAMP and oATP. Complete inactivation was correlated with the binding of about 11 mol [14C]oADP/mol F1. After correction for non-specific labeling, the number of specifically bound [14C]oADP was 2–3 mol per mol F1. By SDS-polyacrylamide gel electrophoresis, [14C]oADP was found to bind covalently mainly to the α and β subunits. In the presence of Mg2+, oATP behaved as a substrate and was slowly hydrolyzed.

Possible interactions between chloroplast and mitochondria remain poorly known, particularly in response to stress conditions. Under these conditions, it is suggested that the electron transfer pathways linked to AOX or pUCP (mitochondrial uncoupling proteins) and PTOX (plastidial uncoupling protein) could limit the formation of ROS to reduce oxidative damage in cellular organelles. In our work, we selected two cultivars of Vigna unguiculata, cv 1183 and cv EPACE. Under our experimental conditions, both cultivars showed no real differences in sensitivity to drought. However cv EPACE was more tolerant to O3 based on the development of necrosis and several physiological parameters (Fv/Fm, [phi]PSII) and biochemical (glutathione content). For the expression profiles of genes encoding AOX, PUCP and PTOX two responses were clearly identified in the cultivar EPACE. On a shortterm, expression of these proteins is generally stimulated. On a longer term (14 days), the answer differs depending on the treatment. Under O3, the strongest expression of mitochondrial proteins is maintained while the gene encoding the PTOX is down-regulated. Under drought only the plastid protein (PTOX) is upregulated. Under conditions of stress combination, drought has little effect on the influx of O3 in the leaves, and the VuPTOX and VuUCP1b genes are up-regulated after 3 and 7 days of stress. This over-regulation, already observed in response to drought alone could play a role in preventing the formation of ROS in both mitochondria and chloroplasts, Les interactions entre chloroplastes et mitochondries restent encore mal connues, notamment en réponse à des conditions de stress. Dans ces conditions, il est suggéré que les voies de transfert d'électrons liées à des protéines découplantes AOX ou pUCP (mitochondriales) et PTOX (plastidiale) pourraient limiter la formation de ROS afin d'atténuer les dommages oxydatifs dans ces organites. Dans le cadre de notre travail, nous avons retenu deux cultivars de Vigna unguiculata, cv 1183 et cv EPACE. Les deux cultivars n'ont pas montré de réelles différences de sensibilité à la sécheresse. Par contre le cultivar EPACE s'est montré plus tolérant à l'O3 sur la base du développement des nécroses et plusieurs paramètres physiologiques (Fv/Fm, [phi]PSII) et biochimiques (glutathion). Pour les profils d'expression des gènes codant pour l'AOX, la pUCP et la PTOX deux réponses ont été clairement identifiées chez le cultivar EPACE. Sur un court terme l'expression de ces protéines est généralement stimulée. Sur un plus long terme (14 jours), la réponse diffère en fonction de la contrainte. Sous O3, la plus forte expression des protéines mitochondriales est maintenue alors que le gène codant pour la PTOX est sousrégulé. Sous sécheresse seule la protéine plastidiale (PTOX) reste sur-régulée. Dans des conditions de combinaison de contrainte, la sécheresse a peu d'effet sur l'influx d'O3 dans les feuilles, et les gènes VuPTOX et VuUCP1b sont sur-régulés après 3 et 7 jours de contrainte. Cette sur-régulation, déjà observée en réponse à la sécheresse seule, pourrait jouer un rôle déterminant pour prévenir la formation de ROS à la fois dans la mitochondrie et dans le chloroplaste