Os metais pesados são elementos químicos com alta densidade (>5 g.cm-3), presentes naturalmente na crosta terrestre, e o aumento das atividades antropogênicas vêm acelerando a liberação destes elementos nos ecossistemas. Tais elementos podem ser extremamente tóxicos (ex. mercúrio, chumbo, cádmio) ou tóxicos quando em concentrações elevadas (zinco e cobre). A fitorremediação é uma técnica de descontaminação que utiliza as plantas para remover poluentes do ambiente ou transformá-los em formas menos perigosas para os seres vivos, sendo que o impacto ambiental e custos de implementação são muito inferiores àqueles alcançados pelos métodos físico-químicos. Uma cultura para ser utilizada com sucesso na recuperação de áreas contaminadas, deve ser eficiente na acumulação de metais, produzir grandes quantidades de biomassa e ser capaz de se adaptar as condições do ambiente contaminado. A cana-de-açúcar apresenta características fisiológicas, que a tornaram uma das culturas mais importantes e difundidas nas regiões tropicais, com alta produtividade de biomassa e rusticidade, possuidora de genes que codificam peptídeos quelantes de metais, já identificados no projeto genoma da cana-de-açúcar (SUCEST). Neste trabalho foi estudada a expressão gênica de metalotioneínas em plântulas de canade- açúcar. As plântulas foram cultivadas em hidroponia contendo níveis elevados de Cu+2 (50, 100, 250 e 500 µM) e Cd+2 (100, 250 e 500µM). A concentração do metal foi determinada nos tecidos de raiz e partes aéreas de plantas coletadas após 11 e 33 dias de tratamento. As maiores doses de Cu (250 e 500 µM) foram letais para as plantas após 11 dias de cultivo, entretanto o efeito das menores dosagens no crescimento foi comparável ao controle aos 33 dias de tratamento. As plantas foram capazes de acumular quantidades significativas de cobre e cádmio, principalmente nas raízes. Todas as dosagens de cádmio limitaram significativamente o crescimento, mas não o impediram. Extratos de RNA total foram analisados para a expressão dos genes de metalotioneína dos tipos I, II e III mediante Northern blot. Os resultados mostraram que houve expressão dos genes para metalotioneínas dos três tipos constitutivamente, sendo mais elevada para MT I e MT II, tanto em folhas como em raízes de cana de açúcar. A expressão de MT I foi maior nas raízes que nas folhas. Já os genes MT II e MT III foram mais expressos em folhas que em raízes. Os genes de metalotioneínas de cana-deaçúcar são pouco afetados ou não são modulados por Cobre. Em relação ao Cd, ocorre um acúmulo de transcritos de MT nas raízes aos 11 dias levemente proporcional à concentração do metal, (exceto para MT III) que é seguido de um decréscimo de transcritos, também paralelo aos teores de Cd na solução, aos 33 dias. Da mesma forma, existe uma indução de MT II e principalmente MT III na parte aérea, com o aumento da concentração de cádmio na solução, mais acentuada aos 11 dias. Estes resultados evidenciam a capacidade da cana-de-açúcar de tolerar e acumular Cu e Cd, sugerindo um potencial fitorremediador para recuperação de áreas contaminadas com esses metais. / Heavy metals are elements with high density (>5 g.cm-3), naturally occurring in soils, and with the increase of anthropogenic activities has accelerated the release of these elements in ecosystems. Heavy metals can be highly toxic (e.g. Mercury; Lead; Cadmium) or toxic at high concentration (Zinc and Copper). Phytoremediation is a clean up technology based on the use of plants to remove pollutants from the environment or to transform them in less toxic forms to living organisms, with less environmental impact at a lower cost than physic-chemical methods. A crop to be successfully used to recover contaminated areas must be efficient in metal accumulation, must yield large amounts of biomass and to be able to adapt to contaminated conditions. Sugarcane (Saccharum spp.) presents physiological characteristics that allowed this crop to become widely adopted in tropical, with high biomass production and with genes associated with chelating peptides already identified by the Sugarcane genome project (SUCEST). In this work, metallothionein gene expression was investigated in sugarcane plantlets. The young plants were growth in ¼ Hoagland solution containing elevated levels of Cu+2 (50, 100, 250 and 500 µM) or Cd+2 (100, 250 and 500µM). Metal concentration were determined in root and shoot tissues harvested at 11 and 33 days of treatment. The highest Cu doses (250 and 500 µM) killed the plants after 11 days of culture, however the effect of the lowest dosages were comparable to the control after 33 days of treatment. The plants were capable of accumulate large amount of Cu and Cd, mainly in roots. All Cd dosages significantly limited growth, but without stopping it. Total RNA was analyzed for metallothionein Type I, II and III gene expression by northern. Results indicated that expression of the three types of metallothionein genes was constitutive, with more expression of MT I and MT II, in leaves and roots. MT I expression was greater in roots than in shoots. But MT II and MT III genes were more expresses in shoots. Metallothionein gene expression was poorly affected or not at all by Cu. But for Cd, there was an accumulation of MT transcripts in roots at 11 days slightly proportional to Cd concentration, except for MT III, which was followed by a decrease in transcripts, also parallel to Cd levels in solution at 33 days. Similarly, there as an induction of MT II and mainly MT III in shots with the increase of Cd concentration in solution, more pronounced at 11 days of treatment. These results demonstrated the ability of sugarcane to tolerate and accumulate Cu and Cd, suggesting a potential application in phytoremediation of areas contaminated with heavy metals.

Desde a década de 90, uma nova família de moléculas de origem protéica e com características hormonais vem sendo estudada em plantas. Esse grupo de novas moléculas, coletivamente chamado de peptídeos hormonais, está envolvido com defesa, reprodução, crescimento e desenvolvimento. RALF, que é um desses novos peptídeos, é ubíquo em plantas e está envolvido com desenvolvimento vegetal. Em Arabidopsis existem 34 genes semelhantes ao RALF (AtRALFs). Nosso grupo mostrou que AtRALF1 é processado de um precursor maior por uma subtilase. Arabidopsis possui 56 subtilases, o objetivo deste trabalho é identificar a subtilase responsável pelo processamento do AtRALF1. Predição da localização subcelular e análise da expressão in silico, ambas confirmadas por análise da expressão por RT-PCR e fusões proteicas com a proteína verde fluorescente, permitiram reduzir para sete o número de subtilases candidatas. Cruzamentos entre mutantes nocaute ou plantas de baixa expressão por RNAi dessas sete subtilases com plantas superexpressoras do AtRALF1 identificaram as subtilases AtSBT6.1 (At5g19660) e AtSBT5.3 (At2g04160) como prováveis envolvidas no processamento do prepropeptídeo AtRALF1. / Since the 90s, a new family of molecules of protein origin and with hormone characteristics has been studied in plants. This group of new molecules, collectively named peptide hormones, is involved in defense, reproduction, growth and development. RALF, one of these peptides, is ubiquitous in plants and is involved in plant development. In Arabidopsis there are 34 RALF-like genes (AtRALFs). Our group has shown that AtRALF1 is processed from a larger precursor by a subtilase. Arabidopsis has 56 subtilases, our goal is the identification of the specific subtilase that is responsible for the AtRALF1 processing. Prediction of subcelular localization and in silico gene expression analysis, both confirmed by RT-PCR expression analysis and chimeric proteins with green-fluorescent protein, allowed the reduction of the initial 56 candidates to only 7 subtilases. Crosses between knockout mutants or RNAi plants expressing low levels of subtilases with overexpressors of AtRALF1 identified the subtilases AtSBT6.1 (At5g19660) and AtSBT5.3 (At2g04160) as potentialy involved in the prepropeptide AtRALF1 processing.

A bacteriose, ou mancha oleosa, doença causada por Xanthomonas axonopodis pv. passiflorae, é um sério problema em muitas áreas de produção de maracujá no Brasil, especialmente se associada à antracnose. A transformação genética é uma alternativa para obter plantas resistentes. Proteínas bactericidas, como as atacinas encontradas na hemolinfa de insetos, têm sido usadas para conferir resistência a espécies vegetais. Como as atacinas têm um peptídeo sinal que as direciona para o espaço extracelular em insetos, nós iniciamos este estudo investigando o direcionamento da atacina A em plantas. A seqüência do gene da atacina A (attA) com e sem o peptídeo sinal foi fusionada com os genes repórteres uidA e gfp e epidermes de cebola foram transformadas, via biobalística, com essas construções gênicas. A atacina A, de fato, é acumulada no apoplasto onde, justamente, bactérias fitopatogênicas se multiplicam antes de invadir as células vegetais. Visando obter plantas transgênicas resistentes à bacteriose, foram transformados tecidos foliares e hipocotiledonares com as linhagens LBA 4404 e EHA 105 de Agrobacterium tumefaciens contendo o gene attA. De um total de 313 explantes infectados, foram obtidos 31 brotos PCR+, o que representa uma eficiência de transformação da ordem de 10%. A expressão do transgene foi confirmada por RT-PCR e a resistência ao patógeno foi avaliada pela inoculação de X. axonopodis pv. passiflorae em folhas destacadas de plantas mantidas in vitro. Em dez plantas não houve formação de lesão foliar, indicando uma possível resistência ao patógeno. / Bacterial spot disease caused by Xanthomonas axonopodis pv. passiflorae is a serious problem in many passion fruit production areas in Brazil, especially if associated with anthracnose. Genetic transformation provides an alternative for obtaining resistant plants. Bactericide proteins such as attacins, found in the haemolymph of insects, have been used to confer resistance on plant species. As the attacins have a sign peptide that dispatches them to extracellular space in insects, we initiated our studies investigating the attacin A directing in plants. The attacin A gene (attA) sequence, with and without the sign peptide, was fused to uidA and gfp reporter genes, and onion epidermis were transformed using bioballistics with gene constructions. The protein did accumulate in the apoplast, where bacteria multiply before attacking plant cells. With the aim of obtaining transgenic plants of yellow passion fruit resistant to bacterial disease, leaf and hypocotyl-derived tissues were transformed with LBA 4404 and EHA 105 strains of Agrobacterium tumefaciens containing the attA gene. From a total of 313 infected explants, we obtained 31 PCR+ shoots, a transformation efficiency of 10%. Expression of the attA gene was confirmed by RT-PCR, and pathogen resistance evaluated by X. axonopodis pv. passiflorae inoculation in leaves obtained from in vitro plants. Leaf lesions were not observed in 10 shoots, suggesting a possible resistance to pathogen.

The increasing environmental concern has reawakened an interest in materials based on renewable resources as replacement for petroleum-based materials. The main objective of this thesis was to explore plant proteins, more specifically wheat gluten, as a binder in wood adhesives intended for typical solid wood applications such as furniture and flooring. Alkaline and acidic dispersions of wheat gluten were used as wood adhesives to bond together beech wood substrates. Soy protein isolate was used as a reference. The tensile shear strengths of the substrates were measured for comparison of bond strength and resistance to cold water. AFM in colloidal probe mode was used to investigate nanoscale adhesion between cellulose and protein films. Wheat gluten was divided into the two protein classes; glutenins and gliadins, and their adhesive performance was compared with that of wheat gluten. Heat treatment and mild hydrolysis were investigated as means for improving bonding performance of wheat gluten. The treated wheat gluten samples were analysed by SE-HPLC and 13C-NMR to correlate molecular size distribution and structural changes with bonding performance. Soy protein isolate is superior to wheat gluten, especially in regards to water resistance. However, the bond strength of wheat gluten is improved when starved bond lines are avoided. The AFM analysis reveals higher interfacial adhesion between soy protein isolate and cellulose than between wheat gluten and cellulose. These results partly explain some of the differences in bonding performance between the plant proteins. Soy protein isolate contains more polar amino acid residues than wheat gluten and possibly interacts more strongly with cellulose. Furthermore, the bond performances of wheat gluten and glutenin are similar, while that of gliadin is inferior to the others, especially regarding water resistance. The extent of penetration of the dispersions into the wood material has a large impact on the results. The bonding performance of gliadin is similar to the others when over-penetration of the dispersion into the wood material is avoided. Moreover, the bond strength of the wheat gluten samples heated at 90°C was in general improved compared to that of wheat gluten. A small improvement was also obtained for some of the hydrolyzed wheat gluten samples (degree of hydrolysis: 0-0.6 %). The improvements in bonding performance for the heat treated samples are due to polymerization, while the improvements for the hydrolyzed samples are due to denaturation. The 13C-NMR analysis of the treated samples confirms some degree of denaturation. / QC 20120514

The transition to flowering is an important decision for plants since seed-setting and the survival of the progeny depend on the environmental conditions prevalent during this transition. Therefore, to ensure maximum reproductive success, plants have evolved several regulatory mechanisms to enable them flower at the most appropriate time. Environmental parameters such as light, temperature and nutrient availability as well as endogenous factors such as age and hormonal status of the plant profoundly affect floral transition (Boss et al., 2004; Srikanth and Schmid, 2011). Studies in Arabidopsis and other model plant species have identified several distinct genetic pathways that integrate the information from the endogenous and environmental cues to regulate flowering (Boss et al., 2004; Srikanth and Schmid, 2011). Many components and gene regulatory networks identified in Arabidopsis are conserved in other commercially important species including rice, maize, sorghum, potato and tomato. Therefore, it is important to understand the basic mechanisms that modulate the flowering response in model plants such as Arabidopsis thaliana, the knowledge from which can be used to develop better adapted and high-yielding varieties of crop plants in the wake of challenges like global warming and increasing food demand. In the present study, we have studied the function of VLB1 and VLB2, genes that code for plant-specific Zn-finger transcription factors. Previous studies from our laboratory (Pratibha Choudhary, Ph.D thesis, 2011) and by other research groups have reported that VLBs redundantly promote flowering in A. thaliana (Yasui et al., 2012; Celesnik et al., 2013). However, the underlying mechanism of this regulation is not well understood. Our data suggests that VLBs redundantly promote the transition to flowering specifically in the photoperiod pathway, the major floral induction pathway in A. thaliana. CO, which is the 93 key regulatory gene in this pathway, is regulated by various factors at the transcriptional as well as post-transcriptional level (Suarez-Lopez et al., 2001; Yanovsky and Kay, 2002; Srikanth and Schmid, 2011). Using genetics, we show that VLBs and CO function together to promote flowering in the photoperiod pathway. Further, our BiFC results reveal that VLBs and CO interact physically. Nevertheless, the physical interaction between VLBs and CO needs to be further validated by in vitro and in vivo by co-immunoprecipitation experiments. We hypothesize that the interaction between VLBs and CO is important to regulate FT expression and hence, flowering. However, whether VLBs interact with CO and promote the CO-stability, or facilitates its recruitment to the FT promoter region, still needs to be determined. Apart from its role in flowering, VLBs have been recently shown to regulate biotic and abiotic responses in Arabidopsis (Nakai et al., 2013a; Nakai et al., 2013b). Also, even though it has been demonstrated that VLBs code for transcription factors, no direct targets of VLBs have been reported till date. We performed a whole genome trancriptome-profiling and found that several important classes of genes including WRKY, RLPs, NBS-LRR and JAZs were affected suggesting that, in addition to their role in floral transition, VLBs have important functions in other plant processes as well. In fact, vlb1vlb2 mutant showed an early senescence phenotype and many senescence-associated genes (SAGs) were up-regulated in our microarray experiments, which was further validated by qRT-PCR analysis. By comparing the differentially-regulated genes and PatMatch analysis, we have identified 82 putative direct targets of VLBs in the Arabidopsis genome which need to be validated by chromatin immunoprecipitation (ChIP) assay and functional studies. 94 Results of global transcriptome analysis revealed that the expression of several JA-signaling and response genes was significantly down-regulated. JA is an important phytohormone involved in plant defense and other developmental processes such as stamen development, root growth and senescence (Wasternack, 2007). Results from the JA-induced expression analysis and root inhibition assay confirmed that JA-signaling and response are indeed compromised in the vlb1vlb2 double mutant. Moreover, in vitro DNA-binding assay showed that MYC2, the key transcriptional regulator of JA-responsive gene expression, is a direct transcriptional target of VLB2. A recent study reported that loss-of-function of VLB genes impairs plant defense while their overexpression confers biotic stress tolerance in Arabidopsis (Nakai et al., 2013a; Nakai et al., 2013b). Compromised JA signaling in the vlb1vlb2 double mutant might partly explain this reduced tolerance to pathogens. However, whether VLBs are associated with the MYC2 promoter in planta needs to be tested by performing ChIP and other in vivo assays. In conclusion, our study shows that VLBs have important regulatory roles in diverse processes including control of flowering time, senescence and JA signaling in Arabidopsis. The validation and functional characterization of the direct targets of VLBs will shed more light on the role of VLBs. Since VLBs are conserved in vascular plants, it will be interesting to see if the function of VLBs is also conserved across species and what might be its ancestral function in evolution.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / This study aimed to evaluate the effects of adding protein sources coming from the pumpkin seed in growth, activity of proteolytic digestive enzymes and metabolic responses of silver catfish (Rhamdia quelen) fed with increasing replacement of fishmeal by pumpkin seed meal (PSM) or phosphorylated protein concentrate of pumpkin seed (PPCPS). For the development of protein concentrate, the method was optimized by statistical methodology of response surface (MRS) and central composite rotational design (CCRD). The CCRD pointed as the highest yield 50.04%, using 4% sodium trimetaphosphate (STMP) at pH 4.5. Those same experimental conditions the protein content was 62.56%. Subsequently, the PSM and PPCPS were used as substitutes in silver catfish protein diets. For eight experimental weeks, 400 silver catfish, with a body mass of 24 ± 0.46 g were divided into 24 fiber glass, fed five experimental diets in four replicates. For the data, four orthogonal contrasts were applied: Control Diet vs. PSM Diets, Control Diet vs. PPCPS Diets, Control Diet vs. Other Diets and PSM Diets vs. PPCPS Diets. Treatment groups replaced 25 to 50 percent protein fishmeal by PSM and PPCPS. Silver catfishes were evaluated for growth parameters, digestive indices, activities of digestive enzymes trypsin and chymotrypsin, as well as metabolic parameters in plasma and liver. The fish growth parameters showed a significant difference (P <0.05) for final body weight at the end of the trial period in contrast Control Diet vs. PSM Diets. Feed conversion was higher for fish that received 25% or 50% PPCPS than those that received PSM (P <0.05). The fish receiving the Control Diet had the lowest digestive-somatic index in contrasts Control Diet vs. PPCPS Diets and Control Diets vs. Other Diets. The control fish treatments showed lower activity of trypsin and chymotrypsin enzymes than fish fed the PSM diet (P <0.05). Also, less chymotrypsin activity was observed in the fish that received the PSM diet (P <0.05), in contrast PSM Diets vs. PPCPS Diets. The fish that received the PPCPS Diets had higher protein efficiency rate than fish fed the FSA (P <0.01). For albumin in plasma, low contents were found in fish that received Control Diet (P <0.05). The ALAT activity was high in fish that received Control Diet (P <0.05), in contrasts Control Diet vs. PSM Diets, Control Diet vs. PPCPs Diets and Control Diet vs. Other Diets. The ALP was most active in fish that received the PPCPS diets (P <0.05), in contrasts Control Diet vs. PPCPS Diets and Control Diet vs. Other Diets. Thus, it can be concluded that the replacement of fishmeal protein by 25 or 50% PPCPS not alter growth and improve the feed conversion catfish and protein efficiency ratio, without affecting the activity of digestive enzymes, metabolic parameters intermediate and liver of silver catfish, can be used in silver catfish nutrition. / O presente estudo teve como objetivo avaliar os efeitos da adição de fontes proteicas advindas da semente de abóbora no crescimento, atividade de enzimas digestivas proteolíticas e respostas metabólicas de jundiás (Rhamdia quelen) alimentados com substituições crescentes de farinha de peixe por farelo de semente de abóbora (FSA) ou concentrado proteico fosforilado de semente de abóbora (CPPFSA). Para o desenvolvimento do concentrado proteico, o método foi otimizado pela metodologia estatística de superfície de resposta (MSR) e delineamento central composto rotacional (DCCR), que apontou como maior rendimento 50,04%, utilizando 4% de trimetafosfato de sódio (STMP) em pH 4,5. Cujo conteúdo proteico foi 62,56%. Posteriormente, o FSA e o CPPFSA foram utilizados como substitutos proteicos nas dietas de jundiás. Durante oito semanas experimentais, 400 jundiás, com massa corporal de 24 ± 0,46 g foram distribuídos em 24 caixas, alimentados com cinco dietas experimentais em quatro repetições. Para análise dos dados, quatro contrastes ortogonais foram aplicados: Dieta Controle vs.. Dietas FSA, Dieta Controle vs.. Dietas CPPFSA, Dieta Controle vs.. Demais dietas e Dietas FSA vs.. Dietas CPFFSA. Os grupos de tratamento substituíam 25 e 50 por cento de proteína da farinha de peixe pelo FSA e CPPFSA. Foram avaliados parâmetros de crescimento, índices digestórios, atividades das enzimas digestivas tripsina e quimotripsina, bem como parâmetros metabólicos em plasma e fígado. Os parâmetros de crescimento dos peixes mostraram diferença significativa (P < 0,05) para massa corporal final ao final do período experimental no contraste Controle vs. dietas FSA. A conversão alimentar foi superior para os peixes que receberam 25% ou 50% CPFFSA em comparação àqueles que receberam FSA (P < 0,05). Os peixes que receberam a dieta Controle apresentaram menor índice digestivo-somático nos contrastes Controle vs. dietas CPFFSA e Controle vs. demais dietas. A atividade das enzimas tripsina e quimotripsina foram menores nos peixes do tratamento Controle, quando comparada aos peixes que receberam as dietas FSA (P < 0,05). Menor atividade de quimotripsina também foi observada nos peixes que receberam as dietas FSA (P< 0,05), no contraste dietas FSA vs. dietas CPFFSA. Os peixes que receberam as dietas CPPFSA apresentaram maior taxa de eficiência proteica do que os peixes que receberam dietas contendo FSA (P< 0.01). Para albumina no plasma, os menores conteúdos foram encontrados nos peixes da dieta Controle (P<0.05). A atividade da enzima ALAT foi maior nos peixes que receberam a dieta Controle (P<0.05), nos contrastes dieta Controle vs. dietas FSA, dieta Controle vs. dietas CPFFSA e dieta Controle vs. demais dietas. A ALP apresentou maior atividade nos peixes que receberam as dietas CPFFSA (P<0.05), considerando os contrastes dieta Controle vs. dietas CPFFSA e dieta Controle vs. demais dietas. Dessa maneira, pode-se concluir que a substituição da proteína da farinha de peixe por 25% ou 50% de CPFFSA não altera o crescimento e promove melhoria na conversão alimentar de jundiá e taxa de eficiência proteica, sem prejudicar atividade de enzimas digestivas, parâmetros metabólicos intermediários e hepáticos dos jundiás, podendo ser utilizado na nutrição do jundiá.

Desde a década de 90, uma nova família de moléculas de origem protéica e com características hormonais vem sendo estudada em plantas. Esse grupo de novas moléculas, coletivamente chamado de peptídeos hormonais, está envolvido com defesa, reprodução, crescimento e desenvolvimento. RALF, que é um desses novos peptídeos, é ubíquo em plantas e está envolvido com desenvolvimento vegetal. Em Arabidopsis existem 34 genes semelhantes ao RALF (AtRALFs). Nosso grupo mostrou que AtRALF1 é processado de um precursor maior por uma subtilase. Arabidopsis possui 56 subtilases, o objetivo deste trabalho é identificar a subtilase responsável pelo processamento do AtRALF1. Predição da localização subcelular e análise da expressão in silico, ambas confirmadas por análise da expressão por RT-PCR e fusões proteicas com a proteína verde fluorescente, permitiram reduzir para sete o número de subtilases candidatas. Cruzamentos entre mutantes nocaute ou plantas de baixa expressão por RNAi dessas sete subtilases com plantas superexpressoras do AtRALF1 identificaram as subtilases AtSBT6.1 (At5g19660) e AtSBT5.3 (At2g04160) como prováveis envolvidas no processamento do prepropeptídeo AtRALF1. / Since the 90s, a new family of molecules of protein origin and with hormone characteristics has been studied in plants. This group of new molecules, collectively named peptide hormones, is involved in defense, reproduction, growth and development. RALF, one of these peptides, is ubiquitous in plants and is involved in plant development. In Arabidopsis there are 34 RALF-like genes (AtRALFs). Our group has shown that AtRALF1 is processed from a larger precursor by a subtilase. Arabidopsis has 56 subtilases, our goal is the identification of the specific subtilase that is responsible for the AtRALF1 processing. Prediction of subcelular localization and in silico gene expression analysis, both confirmed by RT-PCR expression analysis and chimeric proteins with green-fluorescent protein, allowed the reduction of the initial 56 candidates to only 7 subtilases. Crosses between knockout mutants or RNAi plants expressing low levels of subtilases with overexpressors of AtRALF1 identified the subtilases AtSBT6.1 (At5g19660) and AtSBT5.3 (At2g04160) as potentialy involved in the prepropeptide AtRALF1 processing.

Os metais pesados são elementos químicos com alta densidade (>5 g.cm-3), presentes naturalmente na crosta terrestre, e o aumento das atividades antropogênicas vêm acelerando a liberação destes elementos nos ecossistemas. Tais elementos podem ser extremamente tóxicos (ex. mercúrio, chumbo, cádmio) ou tóxicos quando em concentrações elevadas (zinco e cobre). A fitorremediação é uma técnica de descontaminação que utiliza as plantas para remover poluentes do ambiente ou transformá-los em formas menos perigosas para os seres vivos, sendo que o impacto ambiental e custos de implementação são muito inferiores àqueles alcançados pelos métodos físico-químicos. Uma cultura para ser utilizada com sucesso na recuperação de áreas contaminadas, deve ser eficiente na acumulação de metais, produzir grandes quantidades de biomassa e ser capaz de se adaptar as condições do ambiente contaminado. A cana-de-açúcar apresenta características fisiológicas, que a tornaram uma das culturas mais importantes e difundidas nas regiões tropicais, com alta produtividade de biomassa e rusticidade, possuidora de genes que codificam peptídeos quelantes de metais, já identificados no projeto genoma da cana-de-açúcar (SUCEST). Neste trabalho foi estudada a expressão gênica de metalotioneínas em plântulas de canade- açúcar. As plântulas foram cultivadas em hidroponia contendo níveis elevados de Cu+2 (50, 100, 250 e 500 µM) e Cd+2 (100, 250 e 500µM). A concentração do metal foi determinada nos tecidos de raiz e partes aéreas de plantas coletadas após 11 e 33 dias de tratamento. As maiores doses de Cu (250 e 500 µM) foram letais para as plantas após 11 dias de cultivo, entretanto o efeito das menores dosagens no crescimento foi comparável ao controle aos 33 dias de tratamento. As plantas foram capazes de acumular quantidades significativas de cobre e cádmio, principalmente nas raízes. Todas as dosagens de cádmio limitaram significativamente o crescimento, mas não o impediram. Extratos de RNA total foram analisados para a expressão dos genes de metalotioneína dos tipos I, II e III mediante Northern blot. Os resultados mostraram que houve expressão dos genes para metalotioneínas dos três tipos constitutivamente, sendo mais elevada para MT I e MT II, tanto em folhas como em raízes de cana de açúcar. A expressão de MT I foi maior nas raízes que nas folhas. Já os genes MT II e MT III foram mais expressos em folhas que em raízes. Os genes de metalotioneínas de cana-deaçúcar são pouco afetados ou não são modulados por Cobre. Em relação ao Cd, ocorre um acúmulo de transcritos de MT nas raízes aos 11 dias levemente proporcional à concentração do metal, (exceto para MT III) que é seguido de um decréscimo de transcritos, também paralelo aos teores de Cd na solução, aos 33 dias. Da mesma forma, existe uma indução de MT II e principalmente MT III na parte aérea, com o aumento da concentração de cádmio na solução, mais acentuada aos 11 dias. Estes resultados evidenciam a capacidade da cana-de-açúcar de tolerar e acumular Cu e Cd, sugerindo um potencial fitorremediador para recuperação de áreas contaminadas com esses metais. / Heavy metals are elements with high density (>5 g.cm-3), naturally occurring in soils, and with the increase of anthropogenic activities has accelerated the release of these elements in ecosystems. Heavy metals can be highly toxic (e.g. Mercury; Lead; Cadmium) or toxic at high concentration (Zinc and Copper). Phytoremediation is a clean up technology based on the use of plants to remove pollutants from the environment or to transform them in less toxic forms to living organisms, with less environmental impact at a lower cost than physic-chemical methods. A crop to be successfully used to recover contaminated areas must be efficient in metal accumulation, must yield large amounts of biomass and to be able to adapt to contaminated conditions. Sugarcane (Saccharum spp.) presents physiological characteristics that allowed this crop to become widely adopted in tropical, with high biomass production and with genes associated with chelating peptides already identified by the Sugarcane genome project (SUCEST). In this work, metallothionein gene expression was investigated in sugarcane plantlets. The young plants were growth in ¼ Hoagland solution containing elevated levels of Cu+2 (50, 100, 250 and 500 µM) or Cd+2 (100, 250 and 500µM). Metal concentration were determined in root and shoot tissues harvested at 11 and 33 days of treatment. The highest Cu doses (250 and 500 µM) killed the plants after 11 days of culture, however the effect of the lowest dosages were comparable to the control after 33 days of treatment. The plants were capable of accumulate large amount of Cu and Cd, mainly in roots. All Cd dosages significantly limited growth, but without stopping it. Total RNA was analyzed for metallothionein Type I, II and III gene expression by northern. Results indicated that expression of the three types of metallothionein genes was constitutive, with more expression of MT I and MT II, in leaves and roots. MT I expression was greater in roots than in shoots. But MT II and MT III genes were more expresses in shoots. Metallothionein gene expression was poorly affected or not at all by Cu. But for Cd, there was an accumulation of MT transcripts in roots at 11 days slightly proportional to Cd concentration, except for MT III, which was followed by a decrease in transcripts, also parallel to Cd levels in solution at 33 days. Similarly, there as an induction of MT II and mainly MT III in shots with the increase of Cd concentration in solution, more pronounced at 11 days of treatment. These results demonstrated the ability of sugarcane to tolerate and accumulate Cu and Cd, suggesting a potential application in phytoremediation of areas contaminated with heavy metals.

abstract: Flaviviruses (FVs) are among the most medically important arboviruses of the world with the Dengue virus (DENV) accounting for a large percentage of infections observed in tropical and subtropical regions of the world. Globalization, travel, and the expanding range of mosquito vectors, such as Aedes aegypti, have increased the potential of infection rates and illnesses associated with FVs. The DENV and the Zika (ZIKV) FVs frequently co-circulate and generally cause mild self-liming febrile illnesses. However, a secondary infection with a heterologous DENV serotype may lead to life threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DHF/DSS have been linked to antibody dependent enhancement of infection (ADE), a phenomenon that occurs when antibodies (Abs) formed against an initial infection with one serotype of DENV cross-reacts but does not neutralize a heterologous DENV serotype in a secondary infection. Furthermore, Abs raised against the ZIKV have been observed to cross-react with the DENV and vice versa, which can potentially cause ADE and lead to severe DENV disease. The ZIKV can be transmitted vertically and has been linked to devastating congenital defects such as microcephaly in newborns. FDA approved treatments do not exist for DENV and ZIKV illnesses. Thus, there is a need for safe and effective treatments for these co-circulating viruses. Here, a tetravalent bispecific antibody (bsAb) targeting the ZIKV and all four serotypes of the DENV was expressed in the Nicotiana benthamiana (N. benthamiana) plant. Functional assays of the DENV/ZIKV bsAb demonstrated binding, neutralization, and a significant reduction in ADE activity against both the DENV and the ZIKV. A single chain variable fragment (scFv) and a diabody based on an antibody directed against the immune checkpoint inhibitor PD-L1, were also expressed in N. benthamiana leaves. The smaller sizes of the scFv and diabody confers them with the ability to penetrate deeper tissues making them beneficial in diagnostics, imaging, and possibly cancer therapy. The past few decades has seen long strives in recombinant protein production in plants with significant improvements in production, safety, and efficacy. These characteristics make plants an attractive platform for the production of recombinant proteins, biologics, and therapeutics. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2019

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