Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-09-04T15:54:50Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_breno_xavier_goncalves.pdf: 1124189 bytes, checksum: 716ee774eb33d8617a69335350693c80 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-09-13T17:56:24Z (GMT) No. of bitstreams: 2 dissertacao_breno_xavier_goncalves.pdf: 1124189 bytes, checksum: 716ee774eb33d8617a69335350693c80 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-09-13T17:56:39Z (GMT) No. of bitstreams: 2 dissertacao_breno_xavier_goncalves.pdf: 1124189 bytes, checksum: 716ee774eb33d8617a69335350693c80 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-09-13T17:57:02Z (GMT). No. of bitstreams: 2 dissertacao_breno_xavier_goncalves.pdf: 1124189 bytes, checksum: 716ee774eb33d8617a69335350693c80 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-01-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Maçãs da cultivar Gala são caracterizadas por processo de maturação/amadurecimento climatérico, ou seja, para haver a transição do estádio pré-climatérico para o amadurecimento, a maioria dos atributos de qualidade sensorial (textura, síntese de aromas e pigmentos, redução da acidez) são dependentes ou parcialmente dependentes do etileno. Nesse contexto, buscou-se identificar proteínas diferentemente acumuladas durante a transição da maturação para o amadurecimento, em condições que inibem ou não a ação do etileno. Para isso, utilizaram-se maçãs, cv. Gala, clone Beigent, colhidas no estádio pré-climatérico, que foram mantidas em temperatura ambiente por 12 dias, após a aplicação ou não do 1-MCP. Como variáveis dependentes avaliou-se a firmeza de polpa, o teor de sólidos solúveis totais e acidez total titulável, o acúmulo de transcritos do gene ACCO1, a produção de etileno, e proteínas diferentemente acumuladas. Como esperado, a aplicação de 1-MCP contribuiu para preservar a firmeza de polpa e da acidez total, assim como para reduzir significativamente a produção de etileno, provavelmente pela redução da transcrição do gene ACCO1 (redução de 3,5 vezes nos níveis de transcritos em relação às maçãs que não receberam 1-MCP), e consequente redução da síntese da ACCO1 (16 vezes menos em relação às maçãs que não receberam 1-MCP). Pela análise de proteínas, foram visualizados 200 spots, dos quais, 15 diferentemente expressos. Além das proteínas relacionadas com a biossíntese de etileno e metabolismo primário, foram também identificadas proteínas envolvidas no processo de detoxificação e defesa, bem como proteínas reguladoras, a exemplo da 14-3-3 Family protein. Dessa forma, a técnica de proteômica, além de permitir a identificação de proteínas em rotas clássicas, permite identificar proteínas associadas em novos processos relacionados ao amadurecimento de maças. / Apple fruits (c.v. Gala), are characterized by climateric ripening, thus, to bring about the transition from the pre-climacteric to the maturation stage and produce the most of sensory quality attributes (texture, synthetic flavor and pigments and reduction of acidity), this fruits are ethylene dependent. In this context, we investigated the differently accumulated proteins in the ripening process with ethylene inhibit and with ethylene action not inhibited. For this, we used Gala apples, Beigent, clone collected in pre-climacteric stage and kept at room temperature for 12 days after application or not of 1-MCP. As dependent variables we evaluated firmness, total soluble solids, titratable acidity, transcripts accumulation of ACCO1 gene, ethylene production and the differently accumulated proteins. As expected, the application of 1-MCP contributed to maintain the firmness and the total acidity as well as significantly reduce of ethylene production, probably by reducing the transcription of ACCO1 gene (3.5-fold compared to apples without MCP-1), and consequently decrease synthesis of ACCO1 protein (16-fold compared to apple fruits without 1-MCP). We found 200 spots, which 15 differently expressed. We found proteins related to the ethylene biosynthesis and primary metabolism. Further, were also identified proteins involved in detoxification and defense process, as well as regulatory proteins such as 14-3-3 Family Protein. Overall, this proteomic investigation provides a contribution towards better understanding of the regulatory networks involved in fruit ripening process, which could help future studies of apple breeding.

A conversão do amido, armazenado nas frutas durante seu desenvolvimento, em açúcares, é desempenhada por várias enzimas, constituindo-se em um dos principais processos do amadurecimento. A função das enzimas hidrolíticas, alfa-amilase e beta-amilase, no metabolismo amido-sacarose durante o amadurecimento de bananas, foi avaliada através da determinação dos perfis de transcrição e tradução dos seus genes. Utilizando-se da expressão heteróloga de clones de cDNA das amilases, foi possível obter as proteínas recombinantes, inclusive na sua forma enzimaticamente ativa, bem como induzir a produção de anticorpos policlonais em coelhos, com os quais pode-se acompanhar a variação nos níveis de expressão de cada uma das duas enzimas. O tratamento de bananas com o hormônio etileno induziu a antecipação dos processos de degradação do amido e síntese de açúcares em relação ao grupo de frutas controle. Enquanto no grupo controle, as variações nos níveis de proteína e transcrição relativos à alfa-amilase sugerem que há redução na expressão do gene, no grupo tratado com etileno não foi possível detectar a expressão da proteína, apesar dos incrementos na transcrição e atividade. Tal fato pode ser associado à degradação do grânulo de amido e conseqüente solubilização de proteínas ligadas à sua superfície e ao provável aumento no turnover de proteínas promovidos pelo etileno. Em resposta ao tratamento com etileno, houve antecipação dos picos de atividade relacionados especificamente com a beta-amilase, o mesmo ocorreu na detecção do transcrito e sua proteína. Tais resultados sugerem que em bananas, os padrões de expressão e atividade da beta-amilase estão diretamente relacionados à degradação do amido, respondendo também às variações hormonais na fruta, não sendo possível afirmar o mesmo para alfa-amilase. / The starch breakdown in plants is accomplished by several enzymes and pathways and it is the main feature of the ripening in climacteric fruits, such as banana. The function of the hydrolytic enzymes, alpha-amylase and beta-amylase, in the starch-to-sugar metabolism during banana ripening, was evaluated through the determination of the profiles of transcription and translation of its genes. Using the heterologous expression of amylases cDNA clones, was possible to get recombinant proteins in its enzymatically active form, as well as inducing the production of the polyclonal antibodies in rabbits, and use this to evaluate the expression profile of each one enzyme. The treatment of bananas with the hormone ethylene induced the anticipation of the processes of degradation of starch and synthesis of sugars in relation to the control group. While in the control group the variations of protein and transcription levels for alpha-amylase suggests a reduction in the gene expression, in the ethylene group was not possible to detect the expression of the protein, despite the increments in the transcription and activity. Such fact can be associated with the degradation of the starch granules and the resultant surface protein solubilization, and the probable increase in the protein turnover promoted by ethylene treatment. In response to the ethylene, the peak related to beta-amylase activity has been anticipated and the same was occurred with the transcription and translation of this enzyme. These results suggest that the profile of expression and activity of beta-amylase are directly related to the degradation of starch and do respond to hormonal treatment of banana fruits, which could not be affirmed for the enzyme alpha-amylase.

Our knowledge of the factors mediating ethylene-dependent and -independent ripening of climacteric fruit remains limited. Besides the known importance of ethylene roles, auxin has also been emerged as crucial to regulating ripening. Furthermore, the crosstalk between ethylene and auxin in tomato fruit ripening still awaits clarification. ERFs (Ethylene Responsive Factors) are transcription factors belonging to a large family acting downstream on ethylene signaling that direclty regulate ripening-related metabolisms, but their specific roles are still lacking. We present here a comprehensive expression profiling of tomato ERFs in wild-type and tomato ripening-impaired tomato mutants (Nr, rin and nor) indicating that out of the 77 ERFs present in the tomato genome, 27 show enhanced expression at the onset of ripening, while 28 display a ripening-associated decrease in expression, suggesting that different ERFs may have contrasting roles in fruit ripening. Members of subclass E, ERF.E1, ERF.E2 and ERF.E4, show dramatic down-regulation in the ripening mutants suggesting their expression might be instrumental to fruit ripening. The study illustrates the high complexity of the regulatory network interconnecting RIN and ERFs and identifies subclass E members as the most active ERFs in ethylene- and RIN/NOR-dependent ripening. Additionally, with the aim to shed more light into ethylene and auxin interplay, hormonal treatments were applied to tomato fruits and several ripening aspects were then evaluated such as the volatile profile. Overall, results elicited that auxin delay the onset of ripening further showing epistatic effects over the influence of ethylene. Several ripening-related genes, including components of the ethylene signaling, were affected by auxin suggesting potential crosstalk points between the two hormones. Moreover, ethylene appears as potentially part of the auxin regulation through inducing its conjugation. The modulation of hormone levels in tomato fruit throughout ripening can be useful to help designing approaches that both improve fruit quality and extend shelf life. / O conhecimento acerca dos fatores dependentes e independentes de etileno que regulam o amadurecimento de frutos climatéricos é ainda limitado. Além da importância conhecida do etileno, a auxina também tem sido apontada como crucial para o controle do amadurecimento. Mais ainda, há poucos estudos envolvendo o crosstalk entre etileno e auxina em frutos de tomateiro. ERFs (Ethylene Responsive Factors) são fatores de transcrição que atuam nos últimos níveis da via de sinalização de etileno, regulando diretamente metabolismos associados ao amadurecimento. Contudo, seus papéis específicos ainda são desconhecidos. O presente estudo revela o perfil detalhado de expressão de ERFs em tomate selvagem e nos mutantes cujo amadurecimento é comprometido (Nr, rin e nor) indicando que dos 77 ERFs presentes no genoma, 27 apresentam aumento de expressão no início do amadurecimento, enquanto 28 apresentam redução, sugerindo que diferentes ERFs possivelmente têm papéis distintos na regulação do amadurecimento. Membros da subclasse E, ERF.E1, ERF.E2 e ERF.E4, apresentam drástica redução de expressão nos mutantes Nr, rin e nor apontando que tais fatores devem atuar fortemente no amadurecimento. O estudo ilustra também a complexidade das vias de regulação envolvendo RIN e ERFs e ainda aponta os membros da subslasse E como os mais ativos ERFs atuando nas vias etileno-dependentes e RIN/NOR-dependentes. Indo além, com o objetivo de se aprofundar no crosstalk entre etileno e auxina, tratamentos hormonais foram aplicados em frutos de tomateiro e diversos parâmetros do amadurecimento foram avaliados. De uma maneira geral, a auxina retarda o amadurecimento e ainda parece sobrepor os efeitos indutórios do etileno. Genes relacionados ao amadurecimento, incluindo genes relacionados à via de sinalização de etileno, foram afetados pela auxina sugerindo potenciais pontos de crosstalk entre os dois hormônios. O etileno ainda parece regular o metabolismo de auxina no fruto via indução de conjugação pela ativação de GH3s. Compreender o papel dos hormônios no controle da maturação é essencial para o desenvolvimento de tecnologias que visam melhorar a qualidade pós-colheita de frutos.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23526号 / 農博第2473号 / 新制||農||1087(附属図書館) / 学位論文||R3||N5357(農学部図書室) / 京都大学大学院農学研究科農学専攻 / (主査)教授 田尾 龍太郎, 教授 那須田 周平, 教授 中﨑 鉄也 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Thesis (Ph.D. (Plant Production)) -- University of Limpopo, 2022 / In 'Hass' avocado fruit, pre-harvest and postharvest factors affecting exocarp colour change during ripening are vital to maintain the industry’s credibility, competitiveness and profitability. Currently, the South African ‘Hass’ avocado fruit exocarp colour development is affected by pre- and postharvest factors, ultimately, fruit does not develop the required purple colour during ripening. These pre- and postharvest factors must be understood in order to implement strategies that avoid downgrading of South African 'Hass' avocado fruit by lucrative markets due to insufficient purple colour development during ripening. In 'Hass' avocado fruit, exocarp colour development is associated with an increase in anthocyanin synthesis and accumulation during ripening. However, limited information is available regarding factors regulating anthocyanin synthesis and accumulation in 'Hass' avocado fruit during ripening. Therefore, the overall aims of this study were to investigate pre-harvest practices and postharvest treatment that increase exocarp anthocyanin synthesis during ripening. In addition, determine whether exocarp glucose and other antioxidants contribute to 'Hass' avocado fruit exocarp colour development during ripening. In chapter 3, the study looked at how crop load adjustment affects ‘Hass’ avocado fruit exocarp colour development during ripening at three different harvest maturities. The crop load adjustment treatments were applied as: high (100%), moderate (50%) and low (25%) at three harvest times (early, mid- and late). After harvest, fruit were stored at 5.5°C for 28 days, thereafter, ripened at 25°C. The experimental design was carried out as 3 x 3 factorial, arranged in a completely randomized design (CRD) with three replications. The results showed that total anthocyanin and cyanidin 3-O-glucoside concentrations of ‘Hass’ avocados increased following crop load adjustment from normal (100%) to moderate (50%) and low (25%) loads, resulting in improved exocarp colour development during ripening. Furthermore, we discovered that fruit harvested from moderate (50%) and low (25%) crop loads accumulated higher exocarp sugars (D-mannoheptulose and perseitol) at three harvest maturities when compared with high crop load (100%). Moreover, total phenolic concentration of fruit harvested from moderate (50%) and low (25%) crop loads was higher than that obtained from high load fruits, irrespective of harvest maturities. In chapter 4, the study examined the interaction between branch girdling and harvest maturation on the development of 'Hass' avocado fruit exocarp colour during ripening. The experimental design was carried out as 2 x 2 factorial, arranged in a completely randomized design (CRD). The results showed that fruit harvested from girdled trees had poor exocarp colour development as compared to fruit harvested from control trees, regardless of harvest time. Fruit harvested from girdled and ungirdled avocado trees did not show significant differences in visual exocarp colour during early and mid-maturity. Apart from crop load adjustment and girdling as pre-harvest methods to manipulate postharvest exocarp colour, glucose was also infused through the pedicel. Studies on the effect of glucose infusion through the pedicel on the exocarp colour of the ‘Hass’ avocado fruit during ripening were presented in chapter 5. The study included five treatments; control fruit with pedicel and infused with distilled water and glucose concentrations (0.05, 0.13 and 0.28 mM). The distilled water, glucose infused and control fruit were stored at 5.5°C for up to 28 days. After cold storage, fruit were kept at ambient temperature 25°C for ripening. The experiment was conducted as a completely randomized design (CRD) with three replications per treatment. The results showed that glucose infusion through the pedicel markedly increased anthocyanin and cyanidin 3-O-glucoside concentration during ripening. Interestingly, glucose concentrations (0.05 and 0.13 mM) resulted in purple colour development after 8 days at 25°C when compared with control, distilled water and highest concentration (0.28 mM). In chapter 6, the relationship between 'Hass' avocado fruit size, exocarp colour and related pigments with antioxidants capacity and sugar concentration during ripening were investigated. The fruit were categorized by their weight; small (< 200 g) and large (> 201 g). Their diameter and length were also measured using a vernier calliper. Fruit ware stored at 5.5°C for 28 days, then ripened at 25°C. The experimental design was carried out as a completely randomised design (CRD), using 25 fruit replications per category. The results showed that small-sized fruit developed the desirable purple to black exocarp colour when compared with large-sized fruit. Additionally, the results showed that small-sized fruit had higher antioxidant capacity as measured by 2,2 diphenyl 1 picrylhydrazyl (DPPH), ascorbic acid and flavonoid content during ripening when compared with large-sized fruit. Furthermore, it was found that small-sized fruit accumulated higher exocarp and seeds (D-mannoheptulose, perseitol, sucrose and glucose) sugar concentration. We demonstrated and concluded that exocarp colour, pigments, antioxidants and sugar concentration are closely related to size in 'Hass' avocado fruit. Knowledge from this thesis contributes toward the understanding of pre and postharvest factors that may influence colour development of ‘Hass’ avocado fruit during ripening. This study contributes towards bridging the gap in the literature on the biochemical changes associated with colour development of ‘Hass’ avocado fruit during ripening / Agricultural Research Council-Tropical and Subtropical Crops (ARC-TSC) and National Research Foundation (NRF)

Thesis (MSc)--Stellenbosch University, 1964. / ENGLISH ABSTRACT: The influence of manganese sulphate sprays on the yield and ripening of fruit of manganese-deficient Vitis vinifera L. (cv. Pinotage) plants was investigated. Ripening was determined in terms of changes in concentration of the indivi= dual and total sugars and organic acids. Increasing concentrations of manganese sulphate resulted in significant increases in the manganese content of the leaves. The higher levels of manganese were associated with an increase in yield. Ripening, however, was retarded, in that the percentage sugar of the fruit was decreased and titrable acid content increased. The principal sugars present in the fruit were sucrose, glucose, fructose and xylose. Malic and tartaric acids were the main organic acid constituents. Glucose and fructose increased sharply, and sucrose and xylose slightly during ripening of the fruit, whereas malic and tartaric acid de= creased. Glucose, fructose, tartaric and malic acid tended to increase with increasing manganese content. Sucrose and xylose were not appreciably affected. An application of 1% manganese sulphate can be recommend- · ed for manganese deficient vineyards, as it results in an increased yield, in addition to delaying ripening until a more favourable time for handling. Furthermore, the lowered sugar content of the fruit may be advantageous for the pro= duction of dry wines from Pinotage grapes . / AFRIKAANSE OPSOMMING: Die invloed van mangaansulfaatbespuiting op die opbrengs en vrugrypwording van Vitis vinifera L. cv. Pinotage, wat aan ernstige mangaantekorte gely het, is ondersoek. Rypwording is met betrekking tot veranderings in die konsentrasies van totale en indiwiduele suikers en sure bepaal. 2. Toenemende konsentrasies Mnso4 (van O.2% tot l.O%) het n betekenisvolle toename in die mangaangehalte van die blare (van 3 tot 80 d.p.m.) tot gevolg gehad. Die ver= hoogde mangaangehalte het gepaard gegaan met n toe= name in opbrengs, terwyl rypwording vertraag is, deurdat die suikerpersentasie en die titreerbare suurgehalte ver= meerder is in vergelyking met die kontroleplante. 3. Die vernaamste suikers in die vrugte was sukrose, glukose, fruktose en xilose. Glukose en fruktose het vinnig en sukrose en xilose geleidelik toegeneem met die verloop van rypwording. In die ryp vrugte was glukose en fruk= tose oorheersend. Glukose en fruktose was geneig om toe te neem met toenemende mangaangehalte, terwyl sukrose en xilose nie beinvloed is nie. 4. Appelsuur en Wynsteensuur was die oorwegende sure in die vrugte. Beide hierdie sure het gedurende rypwording verminder. Hulle konsentrasies het oor die algemeen toegeneem namate die mangaankonsentrasie verhoog is. 5. Dit is afgelei dat bespuiting met 1.0% MnS04 aanbeveel kan word vir wingerde met mangaantekorte, daar dit n toename in opbrengs, gepaard met 'n vertraging in rypwording tot gevolg gehad het. So 'n vertraging mag moontlik in parstyd voordelig wees. Die verlaagde suikerpersentasie van die vrugte is moontlik gunstig vir die bereiding van droe wyne, waarvoor Pinotage hoofsaaklik gebruik word.

La méthylation de l'ADN génomique est l'un des principaux mécanismes épigénétiques qui conduisent à des changements stables et héréditaires de l'expression des gènes sans que cela s’accompagne de la modification de la séquence d'ADN sous-jacente. Elle fait référence à l'addition d'un groupement méthyl sur le carbone 5 des cytosines (5meC). Ces dernières années, l’étude des mécanismes régulant la mise en place et le maintien de de cette méthylation est devenu un thème de recherche importante, en raison de son rôle essentiel dans la régulation du fonctionnement du génome des plantes et des mammifères. La distribution des 5meC sur l’ensemble du génome d’un organisme, encore appelé méthylome, peut être déterminée par différentes méthodes dont le séquençage de l’ADN génomique après traitement au bisulfite de sodium (WGBS ou méthyl C séq). Chez les végétaux, la méthylation de l’ADN peut se produire dans tous les contextes de séquence incluant les motifs symétriques CG et CHG et le contexte dissymétrique CHH (H pouvant être A, T ou C). En fonction du contexte de séquence, la méthylation des cytosines est mise en place et maintenue par trois types différents d'ADN méthyltransférase. [ ] Chez la plante-modèle Arabidopsis, la déméthylation active de l'ADN joue un rôle essentiel dans l'empreinte maternelle et la déméthylation l’ADN génomique lors du développement de l’albumen, mais elles ne semblent pas jouer de rôle essentiel pendant le développement de la plante chez cette espèce. La méthylation de l’ADN génomique peut aussi être perdue après la réplication de l’ADN, lorsque les mécanismes devant assurer son maintien ne sont pas actifs. On parle alors de déméthylation passive de l’ADN génomique. [ ] En conclusion, les observations présentées dans ce travail fournissent un cadre de travail permettant d’analyser les mécanismes moléculaires responsables de la déméthylation de l'ADN se produisant pendant la maturation des fruits de tomate. Ici, nous présentons une analyse complète des conséquences d’une réduction de l’expression du gène de SlDML2 sur le trancriptome et le métabolome des fruits, tout au long de leur développement. La corrélation entre les profils d’expression de gènes réalisées lors de ce travail ( variété WVA106) et les changements de la distribution de la méthylation de l’ADN telles que décrites chez la variété Ailsa craig montre qu’en plus d'un rôle général dans la régulation des gènes directement impliqués dans plusieurs voies métaboliques, plusieurs gènes codant pour des facteurs de transcription ainsi que des régulateurs épigénétiques sont également susceptibles d'être directement contrôlés par la méthylation de leur région promotrice. Cependant, nous ne pouvions pas établir une relation stricte entre la diminution de la méthylation de l'ADN et l'induction de l'expression des gènes, car de nombreux gènes présentant une diminution du niveau de méthylation de l'ADN dans leur région promotrice pendant la maturation des fruits sauvages correspondent à des gènes normalement réprimés. Ceci suggère que la méthylation active de l'ADN serait nécessaire à leur répression pendant le processus de maturation. Ainsi la relation entre la déméthylation de l'ADN et l'expression des gènes pourrait être plus complexe et ne se limiterait pas à la simple hypothèse de départ de ce travail: la déméthylation de l'ADN est nécessaire à l'expression de gènes induits au cours de la maturation. La déméthylation active de l'ADN pourrait également être nécessaire à la répression de gènes exprimés uniquement lors des phases précoces du développement des fruits et réprimés lors du murissement. / DNA methylation is one of the epigenetic mechanisms that lead to stable and heritable changes in gene expression without alteration on DNA sequence. DNA methylation refers to the addition of a methyl group to the fifth position of the cytosine ring. In recent years, DNA methylation is becoming more and more widely studied, because of its importance in mammals and plants. Methylated cytosines distribution can be determined across the genome at single-nucleotide resolution, that is methylome, using whole genome bisulfite-sequencing (BS-seq) approaches. [ ] Solanum lycopersicum (tomato) is an important agronomic crop and the main model to study the development and ripening process of climacteric fleshy fruit. Recent studies have now shown that the development and ripening of fleshy fruits relies on the establishment and maintenance of differential transcription patterns and complex regulatory pathways that involve both genetic and hormonal controls are operating at these developmental phases. However, it appears that a full understanding of fruit development and ripening will not be achieved based only on genetic models as suggested by recent studies, which showing an important decrease in global methylation level and demethylation at specific promoters during fruit ripening. [ ] In conclusion, the observations presented in this work provide a framework for analysis of the molecular mechanism of DNA demethylation during fruit ripening of tomato. Here, we provide a comprehensive analysis of the knock down SlDML2 on the trancriptome, metaoblom and DNA methylation in the promoter analysis. The large transcriptional reprogramming that occured in mutant during fruit ripeing was correlated alterations in DNA methylation. Here we highlight the central role of active DNA demethylation during tomato fruit ripening. In addition to a general role in the regulation of genes directly involved in several metabolic pathways, we also found that several transcription factors as well as epigenetic regulators are also likely under direct methylation control. However, we could not establish a district relationship between DNA reduction of DNA methylation and induction of gene expression, as not all DEGs containing a type-a DMRs (decreased DNA methylation during fruit ripening) do not correspond to genes normally induced in WT and repressed in transgenic plants. Some were corresponding to an opposite situation and in a few cases more complex methylation pattern (several DMRs) were also found. Indeed these conclusions are based on methylation analysis obtained in another variety. They might however reflect the situation of WVA106 fruits, although some variations are expectable when the methylome of DML RNAi fruits will be analyzed. Hence the relationship between DNA demethylation and gene expression might be more complex than expected, and not limited to the starting hypothesis of this work: DNA demethylation is an absolute requirement for the expression of critical ripening induced genes. This is indeed clearly in this study, but the analysis presented here also suggest that DNA demethylation might also be necessary for the repression of several genes as well. In addition, from the rencent study in Arabidopsis, ROS1 were found preferentially targets transposable elements (TEs) which are closer to protein coding genes and intergenic regions, which suggesting that ROS1 may prevent DNA methylation spreading from TEs to nearby genes. While in tomato, as our analysis, we found the methylation level of promoter of a number of genes was altered during fruit ripening, therefore, through methylome analysis, we will also get the preference of DNA methylation on TE, this analysis will give us idea that demethylation in fleshy fruit may has other distinct function as it is in Arabidopsis.

O mamão papaia (Carica papaya L.) é uma fruta tropical de grande relevância comercial uma vez que o Brasil é o maior produtor mundial e terceiro maior exportador da fruta. Contudo, por ser uma fruta climatérica, tem uma vida pós-colheita limitada, devido ao rápido Amaciamento da polpa. Neste trabalho foi realizada uma investigação proteômica comparativa de polpa do mamão verde e maduro. Várias centenas de componentes protéicos (spots) foram resolvidos em géis 2-DE (faixa de pH 4-7) utilizando a eletroforese diferencial em gel (DIGE) e posteriormente as imagens geradas foram analisadas pelo programa PDQuest. Os spots diferencialmente expressos foram retirados do gel, digeridos e sequenciados (ESI-Q-TOF-MS/MS). Em geral, as proteínas diferencialmente expressas foram associadas ao metabolismo do etileno, resposta ao estresse, metabolismo de carbono e outros importantes processos fisiológicos. Os papéis de algumas das proteínas identificadas foram discutidos em relação à qualidade dos frutos do mamão. Este estudo fornece a primeira caracterização das mudanças no proteoma da polpa do mamão durante o amadurecimento. Deste modo a identificação de proteínas envolvidas na instalação ou desenvolvimento do amadurecimento pode contribuir para o entendimento deste processo e, também, gerar subsídios para avanços tecnológicos visando à qualidade e diminuição das perdas pós-colheita. / Papaya (Carica papaya L.) fruit is a relevant tropical crop since Brazil is the world\'s largest producer and third largest exporter of fruit. However being a climacteric fruit, has a limited green life due to the rapid pulp softening. We report here a comparative proteomic investigation of fruit pulp from unripe and ripe papayas. Several hundreds of protein components were resolved on 2-DE gels (pH range 4-7) using the differential gel electrophoresis (DIGE) approach, and images were analyzed by PDQuest. The variable components were excised, in-gel digested and were analyzed by ESI-Q-TOF-MS/MS. In general, the differentially expressed proteins were associated to ethylene metabolism, stress response, carbon metabolism and other important physiological processes. The roles of some of the identified proteins were discussed in relation to papaya fruit quality. To the best of our knowledge, this investigation provides the first characterization of the changes in papaya fruit pulp proteome during ripening. Thus the identification of proteins involved in the installation or development of the ripening may contribute to the understanding of this process and also provide subsidies for technological advances aimed at quality and reduction of post harvest losses.

O amadurecimento é um processo complexo e geneticamente programado através do qual o fruto adquire características próprias (níveis de açúcar e acidez, cor, amaciamento, sabor e aroma, entre outras) que o torna atraente aos consumidores. O tomate (Solanum lycopersicum) tem sido largamente utilizado como modelo para os estudos sobre amadurecimento de frutos devido à sua importância nutricional e econômica e aos avanços no entendimento de sua genética e bioquímica. Neste fruto, um conjunto de 20 a 30 substâncias voláteis, entre álcoois, aldeídos, cetonas e ésteres, contribuem para o aroma, sendo elas derivadas principalmente de aminoácidos, ácidos graxos e carotenóides. O hormônio etileno está intimamente relacionado com as alterações metabólicas que ocorrem no amadurecimento, inclusive na geração desses compostos voláteis, através da ativação de fatores de transcrição que regulam genes codificadores de enzimas envolvidas nesse processo. Embora se saiba bastante a respeito da bioquímica que produz compostos de aroma e o envolvimento do etileno nesse processo, pouco se sabe sobre o mecanismo de sua regulação. Além disso, o etileno não é o único regulador do amadurecimento, pois há vários indícios de que as auxinas também estão envolvidas neste processo. Embora crescente, este campo ainda encontra-se pouco explorado quando comparado aos avanços obtidos sobre o papel do etileno no amadurecimento de frutos. Os dados são ainda mais escassos no que se refere a regulação das vias de biossíntese dos compostos voláteis. Assim, este trabalho visa avaliar como a interação entre o ácido indol-3-acético (AIA), a auxina mais abundante em plantas, e o etileno influencia a produção do aroma em frutos de tomateiro. Para tal, tomates da cultivar Micro-Tom foram tratados com AIA e etileno, isoladamente e em conjunto. Os resultados mostraram que os grupos de frutos caracterizaram-se por possuir diferentes perfis de compostos voláteis. Os tratamentos com AIA e etileno+AIA ocasionaram atraso no acúmulo de compostos voláteis derivados de isoprenóides, assim como na transcrição de genes envolvidos na síntese desses compostos: carotenoid cleavage dioxygenases 1A e 1B (S/CCD1A e S/CCD1B). A mudança da cor verde para a vermelha e o acúmulo de licopeno também foram atrasados em resposta a estes dois tratamentos. Foram também avaliados os níveis de transcritos de genes envolvidos na síntese de voláteis derivados de ácidos graxos, sendo eles lipoxigenases (S/LOX), hidroperóxido liases (S/HPL) e álcool desidrogenases (S/ADH), além dos níveis da produção de etileno, e AIA na forma livre e conjugados. Os resultados mostraram-se robustos em relação aos impactos nos perfis de compostos voláteis, causados pelos mesmos tratamentos hormonais, em frutos de tomateiro da variedade Grape. Os dados sugerem que a auxina possui importante papel na formação de compostos voláteis do aroma em frutos de tomateiro, regulando de forma negativa este metabolismo. Esse efeito modulador ocorre provavelmente por meio de interações com o etileno. / Fruit ripening is a complex and genetically programmed process through the fruit acquires characteristics (sweetness and acidity, color, softening, flavor and aroma, etc.) that make it attractive to consumers. The tomato fruit (Solanum lycopersicum) has been widely used as a model for studies on fruit ripening due to its nutritional and economic importance and advances in the understanding of its genetics and biochemistry. A set of 20 to 30 volatile substances, including alcohols, aldehydes, ketones and esters, which were derived from amino acids, fatty acids and carotenoids, contribute to the flavor. The hormone ethylene is closely related to the metabolic changes that occur in the maturation, including the generation of these volatile compounds, through the activation of transcription factors that regulate genes encoding proteins involved in this process. Although the knowledge about the biochemistry pathways that produces flavor compounds and the involvement of ethylene have advanced, little is known about the regulation of this process. In addition, ethylene is not the unique hormone that plays this role on fruit ripening. There is a growing body of evidence indicating the involvement of auxin in the maturation. The role of other hormonal classes is still little explored when compared to progress made on the role of ethylene in fruit ripening, especially regarding the regulation of the biosynthetic pathways of volatile compounds. This study aim to assess how the interaction between the indole-3-acetic acid (IAA), the most abundant auxin in plants, and ethylene influence the production of tomato fruit aroma. To do this, fruit from tomato cultivar Micro-Tom were treated with IAA and ethylene, separately and in combination. The results showed that the fruit groups characterized by having different profiles of volatile compounds. The treatment with IAA and IAA + ethylene caused delay in accumulation of volatile compounds derived from isoprenoid, as well as in the transcription of genes involved in the synthesis of these compounds: carotenoid cleavage dioxygenases 1A and 1B (S/CCD1A and S/CCD1B). The change from green to red and the accumulation of lycopene were also delayed in response to these two treatments. We also assessed the levels of transcripts of genes involved in the synthesis of volatile compounds derived from fatty acids (lipoxygenases [S/LOX], hydroperoxide lyases [S/HPL] and alcohol dehydrogenases [S/ADH]), besides the levels of ethylene production, and IAA in free and conjugated form. The results were robust with respect to impacts on volatile compounds profiles, caused by the same hormone treatments in tomato variety Grape. The data suggest that auxin plays an important role in the synthesis of volatile compounds in tomato fruit, negatively regulating this metabolism. This modulating effect likely occurs through crosstalks with ethylene.

A enzima sacarose-fosfato sintase foi parcialmente purificada de bananas fisiologicamente imaturas (70 dias após a antese), fisiologicamente maturas pré-climatéricas (110 dias após a antese) e climatéricas (130 dias após a antese). De acordo com os resultados apresentados a SPS de banana é uma enzima constituída de subunidade de 116 kD, apresentando peso molecular nativo de 440 kD por filtração em gel e bandas de 180, 240 e 686 kD por eletroforese em gel de poliacrilamida, nos três estágios estudados. Uma sequência parcial do gene da SPS foi amplificado através de PCR, donado e seu sequenciamento indicou que a enzima de banana apresenta elevada homologia com as de outras fontes vegetais. A análise dos níveis de proteina e mRNA durante o desenvolvimento e amadurecimento do fruto permitem correlacionar o aumento de atividade com o aumento na expressão do gene da SPS. Não foram observadas alterações significativas no estado de ativação, sugestivas de modificação covalente como mecanismo de ativação durante o amadurecimento. / Sucrose-phosphate synthase (SPS) was purified ITom bananas at different deveIopmental stages (70, 110 and 130 days after anthesis), corresponding to tissue with different composition. Banana SPS have a subunit of 116 kD and the native enzyme have a molecular weight around 440 kD by gel fiItration and bands of 180, 240 and 686 kD by eIectrophoresis in alI stages studied. A 952 base pair polymerase chain reaction product of SPS was cloned and showed that banana SPS has a high homology with the enzyme from other species. Enzyme activity, Protein and mRNA leveI point to an increase in SPS expression during ripening. The increase in SPS activity was not correlated to changes in the activation state. The results indicate that the increase in SPS expression is an important regulatory event during banana fruit ripening.