This work identifies transcription factors (TF) controlling triacylglycerol (TAG) synthesis and accumulation in plant tissues. TAG plays vital role in plants and are used by humans. Most plants accumulate oil in the seed, but some species accumulate oil in other tissues. The Wrinkled1 (WRI1) TF has been shown to regulate oil accumulation in multiple species and tissues. Here, four WRI homologues in avocado were identified, their phylogeny was examined and three of them were cloned into expression vectors for further characterization. However, WRI1 likely does not act alone in regulation of TAG accumulation in plants. Additional candidate TFs were identified by using transcriptome data from a variety of species, and cloned into expression vectors. Future studies will be able to use this information to better understand regulation of TAG accumulation, which will allow increased oil accumulation in plants for various human uses.

O objetivo deste trabalho foi avaliar a disponibilidade de Fe, Cu e Zn em presença de mesocarpo de babaçu, pois o mesocarpo contem compostos antinutricionais (fitato e lignina) que podem diminuir a disponibilidade de nutrientes minerais. As concentrações de Fe, Cu e Zn encontradas no mesocarpo de babaçu foram 17, 7,0 e 2,6 µg g-1, respectivamente. Para um melhor entendimento das interações dos elementos com o mesocarpo, foi realizada a extração de substâncias complexantes com soluções de NaOH (pH 7 e 12). Análises por espectrometria de absorção molecular indicaram que o principal complexante presente no extrato era o fitato. Soluções de Fe, Cu e Zn (10 a 300 mg L-1) foram adicionadas aos extratos, para que, após agitação e centrifugação, fossem determinadas as recuperações desses elementos. Os resultados mostraram que os elementos formam complexos com o fitato e a solubilidade desses complexos é dependente da razão elemento/fitato, quanto maior essa razão menor é a solubilidade. Também foi avaliada a interação dos elementos com o sólido remanescente da extração. Fe, Cu ou Zn foram adicionados ao mesocarpo lavado. Não foi possível afirmar que houve interação do mesocarpo lavado com Fe e Cu devido à precipitação decorrente da hidrólise. Porém, o Zn não sofreu hidrólise e teve baixas recuperações, indicando que houve interação com o sólido. Além disso, pode ser observado que ao adicionar maiores concentrações de Zn obtiveram-se melhores recuperações, provavelmente devido à saturação dos sítios de ligação. Para os estudos de disponibilidade, foi utilizado o procedimento de digestão simulada in vitro da US Pharmacopeia. Fe e Zn não foram extraídos durante a digestão do mesocarpo, portanto esses elementos não estariam disponíveis para absorção pelo organismo. Entretanto, cerca de 120 µg L-1 de Cu foi extraído do mesocarpo. Foram realizadas digestões do mesocarpo com adição de Fe, Cu e Zn e apenas 48% do Fe, 65% do Cu e 75% do Zn foram recuperados. Digestões contendo essa mesma concentração dos elementos foram realizadas com adições de Ca e Mg. A presença do Ca diminuiu a recuperação de Fe e Zn. Para simular uma situação próxima ao real, foram realizadas digestões gastrointestinais de leite, mistura de leite e mesocarpo e mistura de leite e lignina. Apesar do ferro presente no leite apresentar baixa disponibilidade, quando o mesmo foi misturado ao mesocarpo observou-se um aumento da concentração de Fe solúvel. Compostos presentes no leite ou no mesocarpo podem aumentar a solubilidade dos elementos. Não foi possível observar se havia alguma alteração da disponibilidade do Cu presente no leite na presença do mesocarpo, pois a concentração de Cu no leite é muito baixa. A fração de Zn solúvel na mistura de leite e mesocarpo permaneceu a mesma, porém a fração dialisável foi praticamente nula. A adição de lignina ao leite aumentou a extração dos elementos. Entretanto, esses elementos continuaram não sendo dialisados / The aim of this study was to evaluate the availability of Fe, Cu and Zn in the presence of mesocarp of babassu because it contains antinutritional compounds (phytate and lignin) that may decrease the availability of mineral nutrients. The concentrations of Fe, Cu and Zn found in the mesocarp of babassu were 17, 7.0 and 2.6 mg g-1, respectively. For a better understanding of elements interactions with mesocarp, it was performed the extraction of complexing substances with NaOH solutions (pH 7 and 12). Analysis by molecular absorption spectrometry indicated that the main complexing present in the extract was the phytate. Solutions of Fe, Cu and Zn (10 to 300 mg L-1) were added to the extracts for determining the recoveries of these elements, after agitation and centrifugation. The results showed that the elements form complexes with phytate and the solubility of these complexes is dependent on the ratio element / phytate, the greater this ratio the lower the solubility. The elements interaction with the remaining solid from extraction was also evaluated. Fe, Cu or Zn were added to the washed mesocarp. It was not possible to affirm that there was interaction of washed mesocarp with Fe and Cu due to precipitation derived from hydrolysis. However, Zn did not suffer hydrolysis and had low recoveries, indicating that there was an interaction with the solid. Furthermore, it can be seen that adding higher concentrations of Zn, better recoveries were obtained, probably due to saturation of the binding sites. For the studies of availability, it was used an in vitro simulated digestion procedure from U.S. Pharmacopeia. Fe and Zn were not extracted during digestion of the mesocarp, therefore these elements would not be available for absorption by the organism. However, about 120 mg L-1 of Cu was extracted from the mesocarp. Mesocarp digestions were performed with addition of Fe, Cu and Zn and only 48% of Fe, 65% of Cu and 75% of Zn were recovered. Digestions containing the same concentration of the elements were performed with additions of Ca and Mg. The presence of Ca decreased the recovery of Fe and Zn. For simulating a real situation, gastrointestinal digestion of milk, milk and mesocarp mixture and milk and lignin mixture were performed. Although the iron present in milk had low availability, when it was mixed with mesocarp it was observed an increase in the concentration of soluble Fe. Compounds present in milk or in mesocarp can increase the solubility of the elements. It was not possible to observe if there was any change in the availability of Cu present in milk in the presence of mesocarp, because the Cu concentration in the milk is very low. The fraction of soluble Zn in milk and mesocarp mixture remained the same, but the dialysable fraction was practically null. Lignin addition increases the milk elements extraction. However, these elements remain not dialyzed

O objetivo deste trabalho foi avaliar a disponibilidade de Fe, Cu e Zn em presença de mesocarpo de babaçu, pois o mesocarpo contem compostos antinutricionais (fitato e lignina) que podem diminuir a disponibilidade de nutrientes minerais. As concentrações de Fe, Cu e Zn encontradas no mesocarpo de babaçu foram 17, 7,0 e 2,6 µg g-1, respectivamente. Para um melhor entendimento das interações dos elementos com o mesocarpo, foi realizada a extração de substâncias complexantes com soluções de NaOH (pH 7 e 12). Análises por espectrometria de absorção molecular indicaram que o principal complexante presente no extrato era o fitato. Soluções de Fe, Cu e Zn (10 a 300 mg L-1) foram adicionadas aos extratos, para que, após agitação e centrifugação, fossem determinadas as recuperações desses elementos. Os resultados mostraram que os elementos formam complexos com o fitato e a solubilidade desses complexos é dependente da razão elemento/fitato, quanto maior essa razão menor é a solubilidade. Também foi avaliada a interação dos elementos com o sólido remanescente da extração. Fe, Cu ou Zn foram adicionados ao mesocarpo lavado. Não foi possível afirmar que houve interação do mesocarpo lavado com Fe e Cu devido à precipitação decorrente da hidrólise. Porém, o Zn não sofreu hidrólise e teve baixas recuperações, indicando que houve interação com o sólido. Além disso, pode ser observado que ao adicionar maiores concentrações de Zn obtiveram-se melhores recuperações, provavelmente devido à saturação dos sítios de ligação. Para os estudos de disponibilidade, foi utilizado o procedimento de digestão simulada in vitro da US Pharmacopeia. Fe e Zn não foram extraídos durante a digestão do mesocarpo, portanto esses elementos não estariam disponíveis para absorção pelo organismo. Entretanto, cerca de 120 µg L-1 de Cu foi extraído do mesocarpo. Foram realizadas digestões do mesocarpo com adição de Fe, Cu e Zn e apenas 48% do Fe, 65% do Cu e 75% do Zn foram recuperados. Digestões contendo essa mesma concentração dos elementos foram realizadas com adições de Ca e Mg. A presença do Ca diminuiu a recuperação de Fe e Zn. Para simular uma situação próxima ao real, foram realizadas digestões gastrointestinais de leite, mistura de leite e mesocarpo e mistura de leite e lignina. Apesar do ferro presente no leite apresentar baixa disponibilidade, quando o mesmo foi misturado ao mesocarpo observou-se um aumento da concentração de Fe solúvel. Compostos presentes no leite ou no mesocarpo podem aumentar a solubilidade dos elementos. Não foi possível observar se havia alguma alteração da disponibilidade do Cu presente no leite na presença do mesocarpo, pois a concentração de Cu no leite é muito baixa. A fração de Zn solúvel na mistura de leite e mesocarpo permaneceu a mesma, porém a fração dialisável foi praticamente nula. A adição de lignina ao leite aumentou a extração dos elementos. Entretanto, esses elementos continuaram não sendo dialisados / The aim of this study was to evaluate the availability of Fe, Cu and Zn in the presence of mesocarp of babassu because it contains antinutritional compounds (phytate and lignin) that may decrease the availability of mineral nutrients. The concentrations of Fe, Cu and Zn found in the mesocarp of babassu were 17, 7.0 and 2.6 mg g-1, respectively. For a better understanding of elements interactions with mesocarp, it was performed the extraction of complexing substances with NaOH solutions (pH 7 and 12). Analysis by molecular absorption spectrometry indicated that the main complexing present in the extract was the phytate. Solutions of Fe, Cu and Zn (10 to 300 mg L-1) were added to the extracts for determining the recoveries of these elements, after agitation and centrifugation. The results showed that the elements form complexes with phytate and the solubility of these complexes is dependent on the ratio element / phytate, the greater this ratio the lower the solubility. The elements interaction with the remaining solid from extraction was also evaluated. Fe, Cu or Zn were added to the washed mesocarp. It was not possible to affirm that there was interaction of washed mesocarp with Fe and Cu due to precipitation derived from hydrolysis. However, Zn did not suffer hydrolysis and had low recoveries, indicating that there was an interaction with the solid. Furthermore, it can be seen that adding higher concentrations of Zn, better recoveries were obtained, probably due to saturation of the binding sites. For the studies of availability, it was used an in vitro simulated digestion procedure from U.S. Pharmacopeia. Fe and Zn were not extracted during digestion of the mesocarp, therefore these elements would not be available for absorption by the organism. However, about 120 mg L-1 of Cu was extracted from the mesocarp. Mesocarp digestions were performed with addition of Fe, Cu and Zn and only 48% of Fe, 65% of Cu and 75% of Zn were recovered. Digestions containing the same concentration of the elements were performed with additions of Ca and Mg. The presence of Ca decreased the recovery of Fe and Zn. For simulating a real situation, gastrointestinal digestion of milk, milk and mesocarp mixture and milk and lignin mixture were performed. Although the iron present in milk had low availability, when it was mixed with mesocarp it was observed an increase in the concentration of soluble Fe. Compounds present in milk or in mesocarp can increase the solubility of the elements. It was not possible to observe if there was any change in the availability of Cu present in milk in the presence of mesocarp, because the Cu concentration in the milk is very low. The fraction of soluble Zn in milk and mesocarp mixture remained the same, but the dialysable fraction was practically null. Lignin addition increases the milk elements extraction. However, these elements remain not dialyzed

Plants synthesize and store oil, mostly triacylglycerol (TAG), in various storage tissues that serves as a source of carbon and energy. The process is transcriptionally controlled by WRINKLED1 (WRI1), a member of the APETALA2 (AP2) class of transcription factors, that regulates most of the fatty acid biosynthesis genes. Among the four Arabidopsis WRI1 paralogs, only WRI2 is nonfunctional and failed to complement wri1-1 mutant seeds. The oleaginous Avocado (Persea americana) fruit mesocarp (60-70% DW oil) showed high expression levels for orthologs of WRI2, along with WRI1 and WRI3. While the role of WRI1 as a master seed oil biosynthesis regulator is well-established, the function of WRI1 paralogs in non-seed tissues is poorly understood. We conducted structural analyses to elucidate distinct features of avocado WRI paralogs compared to their orthologs in seed tissues. Comprehensive comparative in silico analyses of WRI1 paralogs from Arabidopsis (dicot), maize (monocot), and avocado revealed distinct features associated with their function. Our analysis showed the presence of only one AP2 domain in all WRI2 orthologs, compared to two AP2 in others. The highly conserved N-terminal region and the less conserved C-terminal regions make up the primary structure of the proteins, with amino acid composition bias characteristic of intrinsically disordered proteins (IDPs). Additionally, the avocado WRI2 showed a high proportion of random coil secondary structure, although it lacks a C-terminal intrinsically disordered region (IDR). Also, both WRI1 and WRI2 have distinct predicted phosphorylation target sites compared to their orthologs, whereas WRI2 lacks a PEST motif. Finally, through transient expression assays, we demonstrated that both avocado WRI1 and WRI2 are functional and drive TAG accumulation in Nicotiana benthamiana leaves. Our study showed that avocado WRI2 is structurally different and is functional, unlike its ortholog in Arabidopsis. This study provides us with new targets to enhance oil biosynthesis in plants.

Avocado is an economically important crop with ~70% oil in its fruit tissue, which is an essential component of human diet. The steady increase in global demand for avocado production (9%/year) has drawn attention to the importance of understanding the genetic regulation of triacylglycerol (TAG) accumulation. Using RNA-seq approach, mesocarp-specific regulation and biosynthesis of TAG in developing avocado fruit was analyzed. During the period of TAG accumulation in the mesocarp, an increased expression was noted for genes mostly associated with hexose metabolism in plastids, including pyruvate kinase, relative to cytosol, which is likely associated with the need for higher pyruvate flux directed toward plastid fatty acid synthesis. A corresponding increase in expression for plastidial fatty acid synthesis genes was also noted but not for TAG assembly genes. Additionally, WRINKLED1 (WRI1), a regulatory element typically associated with seed oil biosynthesis, was also highly expressed in oil-rich mesocarp of avocado, along with two other isoforms of WRI. Transcriptomics also revealed that multiple acyltransferases that participate in rate-limiting step in TAG synthesis might be active concomitantly in mesocarp to achieve higher levels of TAG accumulation. Similar observations were previously made with transcriptome analysis of oil-rich seed and non-seed tissues. Together these data suggest a ubiquitous role for WRI1 and that a major point of regulation of oil biosynthesis in oilrich mesocarp tissue most likely occurs at the level of source and not sink. Overall, this study provides a foundation for functional genomics required to direct metabolic engineering efforts to enhance avocado oil yield.

Plants synthesize and store oil, mostly as triacylglycerols (TAG), in seeds that is transcriptionally controlled by WRINKLED1 (WRI1), an APETALA2 (AP2) transcription factor. Among the four Arabidopsis WRI paralogs, WRI2 is nonfunctional, while the others are expressed in a tissue-specific manner. Additionally, two rate-limiting enzymes, diacylglycerol acyltransferase (DGAT) and phospholipid diacylglycerol acyltransferase (PDAT) catalyze the terminal step in TAG assembly and contribute to oil accumulation. Avocado (Persea americana) mesocarp, a non-seed tissue, accumulates significant amounts of TAG (~70% by dry weight) enriched with heart-healthy oleic acid. The oil accumulation stages in mesocarp coincide with the high expression of PaWRI2, along with PaWRI1, PaDGAT1, and PaPDAT1. The strong preference for oleic acid demonstrated by the avocado mesocarp TAG biosynthetic machinery represents lucrative biotechnological opportunities, yet functional implication of these genes is not explored. Using structural analyses, we showed that PaWRI2 is a relatively stable protein, has a single intact AP2 DNA-binding domain, and has different C-terminal properties compared to its ortholog in Arabidopsis. Through transient expression, we demonstrated that PaWRI2 is functional and drives TAG accumulation in Nicotiana benthamiana leaves, unlike Arabidopsis WRI2. Additionally, co-infiltration of PaWRI2, along with PaWRI1, PaDGAT1, and PaPDAT1 further increased the lipid content and oleic acid levels in ‘benth’ leaves. Quantitative real-time PCR (qPCR) analyses of >46 fatty acid biosynthetic pathway genes revealed that several were distinctly up- or down-regulated by the expression of PaWRI2 and PaWRI1. Further yeast-one-hybrid assay showed a unique characteristic of PaWRI2 being autoregulated and by PaWRI1. Also, both the proteins could bind to AW-box promoter elements in specific avocado genes. Deletion of the C-terminally-located ordered region in both the proteins further improved the lipid content with an altered composition in the leaf tissue. In conclusion, avocado WRI2 is capable of transactivation of fatty acid biosynthesis genes and TAG accumulation, synergistically with DGAT1 and PDAT1, in non-seed tissues. This study provides a functional role for WRI2 in a basal angiosperm species that is likely lost in modern angiosperms and thus provides basis for mechanistic differences in the transcriptional regulation of lipid biosynthesis among different plant species and between seed and non-seed tissues.

Plants are being engineered for enhanced ethanol production; however, challenges remain in meeting the demand for bioenergy that is expected to double by 2030. Therefore, targeting carbon accumulation in the form of energy-dense oils in nonseed biomass is considered a superior alternative for bioenergy production. Although oils in the form of triacylglycerols (TAGs) are typically stored in seed tissues, various nonseed tissues such as mesocarp, tubers, stems and leaves also serve as storage tissues for TAG accumulation in plants. Moreover, the biomass of these tissues is generally far greater than seed biomass. In order to increase oil content in nonseed biomass for bioenergy and nutritional purposes, it is important to understand how such plants naturally accumulate TAG in nonseed tissues. Several molecular approaches, including transcriptomics, have been undertaken to elucidate the metabolic and regulatory mechanisms of carbon partitioning in oil-rich nonseed tissues. Such studies are expected to generate important transgenic tools that can be used to alter fatty acid metabolism and engineer plants to produce oil-rich biomass successfully. This review focuses on the potential of different oil-rich nonseed tissues and the strategies developed for enhancing oil biomass.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2018-11-22T16:49:47Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) ana_claudia_silveira_alexandre.pdf: 1522170 bytes, checksum: ecd371ab3f6777103dd99573de2bf35a (MD5) / Made available in DSpace on 2018-11-22T16:49:47Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) ana_claudia_silveira_alexandre.pdf: 1522170 bytes, checksum: ecd371ab3f6777103dd99573de2bf35a (MD5) Previous issue date: 2018 / Funda??o de Amparo ? Pesquisa do estado de Minas Gerais (FAPEMIG) / A melancia (Citrullus lanatus) ? um fruto refrescante que apresenta versatilidade de consumo e consider?vel potencial nutricional; entretanto, ? respons?vel por uma grande gera??o de res?duos, sendo o mesocarpo (entrecasca), o principal destes. Este res?duo agr?cola subutilizado ? uma boa alternativa para aproveitamento e desenvolvimentos de novos produtos. O presente estudo relata a possibilidade de agrega??o de valor ao mesocarpo de melancia por meio do desenvolvimento de picles acidificados e fermentados. A efici?ncia do emprego de culturas starters, cultura aut?ctone isolada e cultura de Lactobacillus acidophilus foram avaliados e comparados em rela??o ? fermenta??o natural do mesocarpo de melancia. As fermenta??es foram acompanhadas por meio de an?lises f?sico-qu?micas e microbiol?gicas, e os produtos foram caracterizados quanto ? composi??o centesimal, f?sico-qu?mica, qualidade microbiol?gica e sensorial. Inferiu-se que a exist?ncia de bact?rias l?ticas durante os processos fermentativos do mesocarpo de melancia afetou a comunidade microbiana presente, resultando no consumo de carboidratos, produ??o de ?cidos org?nicos e acidifica??o do meio fermentativo. Nas fermenta??es adicionadas de culturas starters, foram alcan?ados menores valores de pH ao final dos processos fermentativos, sendo atingidos pH 3,52 (cultura starter aut?ctone isolada) e pH 3,68 (cultura starter L. acidophilus). A fermenta??o do mesocarpo de melancia resultou em produtos com maior valor nutricional, mais seguros e est?veis. Determinou-se que os picles desenvolvidos foram seguros do ponto de vista microbiol?gico, al?m de apresentarem maior potencial bioativo que o mesocarpo in natura. Os melhores resultados sensoriais foram encontrados para os picles n?o fermentados. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncia e Tecnologia de Alimentos, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2018. / The watermelon (Citrullus lanatus) is a refreshing fruit that presents versatility for consumption and considerable nutritional potential; however, it is responsible for a large generation of residues, the mesocarp (inner rind) being the principal residue. This underutilized agricultural residue is a good alternative for the use and development of new products. The present study reports the possibility of increasing the value to the watermelon mesocarp through the development of acidified and fermented pickles. The efficiency of the use of starter cultures, isolated autochthonous culture and a culture of Lactobacillus acidophilus were evaluated and compared to the natural fermentation of the watermelon mesocarp. The fermentations were accompanied by physicochemical and microbiological analyses, and the products were characterized with regard to the proximate composition, physicochemical characteristics and the microbiological and sensorial qualities. It was inferred that the existence of lactic bacteria during the fermentative processes of the watermelon mesocarp affected the microbial community present, resulting in the consumption of carbohydrates, production of organic acids and acidification of fermentative media. In the fermentations to which starter cultures were added, lower pH values were reached at the end of the fermentation process, reaching pH 3.52 (native starter culture isolated) and pH 3.68 (L. acidophilus culture starter). The fermentation of the watermelon mesocarp resulted in products with higher nutritional value, being more secure and stable. The pickles developed were found to be safe from a microbiological point of view, in addition to having a greater bioactive potential than the fresh mesocarp. The best sensory results were obtained for the unfermented pickles.

No description available.

WRINKLED1 (WRI1), a member of the APETALA2 (AP2) class of transcription factors regulates fatty acid biosynthesis and triacylglycerol (TAG) accumulation in plants. Among the four known Arabidopsis WRI1 paralogs, only WRI2 was unable to complement and restore fatty acid content in wri1-1 mutant seeds. Avocado (Persea americana) mesocarp, which accumulates 60-70% dry weight oil content, showed high expression levels for orthologs of WRI2, along with WRI1 and WRI3, during fruit development. While the role of WRI1 as a master regulator of oil biosynthesis is well-established, the function of WRI1 paralogs is poorly understood. Comprehensive and comparative in silico analyses of WRI1 paralogs from avocado (a basal angiosperm) with higher angiosperms Arabidopsis (dicot), maize (monocot) revealed distinct features. Predictive structural analyses of the WRI orthologs from these three species revealed the presence of AP2 domains and other highly conserved features, such as intrinsically disordered regions associated with predicted PEST motifs and phosphorylation sites. Additionally, avocado WRI proteins also contained distinct features that were absent in the nonfunctional Arabidopsis ortholog AtWRI2. Through transient expression assays, we demonstrated that both avocado WRI1 and WRI2 are functional and drive TAG accumulation in Nicotiana benthamiana leaves. We predict that the unique features and activities of ancestral PaWRI2 were likely lost in orthologous genes such as AtWRI2 during evolution and speciation, leading to at least partial loss of function in some higher eudicots. This study provides us with new targets to enhance oil biosynthesis in plants.