ABSTRACT In the last decades, several biochemical and molecular studies in pear (Pyrus spp.) have revealed processes associated with dormancy establishment and release, leading to the identification of several MADS-box family genes related to bud dormancy regulation. Thus, the aim of this study was to evaluate a preliminary expression pattern of the PpDAM13-1 and PpDAM13-2 MADS-box genes in pear leaf buds (Pyrus pyrifolia Nakai, cultivars Hosui and Nijisseiki; and Pyrus communis L., cultivars Rocha and Santa Maria), grown in the subtropical climate of Southern Brazil. Lateral leaf buds were collected from June to September, and RNA was extracted for evaluation of gene expression by reverse transcription polymerase chain reaction (RT-qPCR). It was found that these two genes decreased the expression as leaf buds are release from dormancy. The Hosui cultivar for both genes and the Nijisseiki cultivar for PpDAM13-2 gene showed peaks of expression in early June, indicating that leaf buds are in a transition dormancy phase. The two European cultivars showed this transition dormancy phase between the end-July and the mid-August. It was concluded that the expression analysis of PpDAM13-1 and PpDAM13-2 genes can be used as a marker to evaluate changes of dormancy phase in pear trees in subtropical climate conditions, and it can be suitable to assist management strategies for overcoming pears’ bud dormancy.

A soja é uma das maiores fontes de proteína vegetal da humanidade. É também muito importante na alimentação animal e como matéria-prima industrial. Grandes avanços foram recentemente alcançados em sua transformação genética. O objetivo desta revisão é fornecer uma discussão acerca de importantes fatores que influenciam a transformação de soja mediada por Agrobaterium, incluindo tecidos alvo, condições do tecido vegetal, métodos de injúria, sistemas de regeneração, marcadores de seleção e genes repórter.

Soybean is one of humanity's major sources of plant protein. It is also very important for animal feed and as industrial raw material. Great advances have recently been achieved in its genetic transformation. This review provides a comprehensive discussion of important factors affecting Agrobacterium-mediated soybean transformation including target tissues, plant tissue health, wounding methods, regeneration systems, selectable markers and reporter genes.

The effects of ethylene on plants have been recognized since the Nineteenth Century and it is widely known as the phytohormone responsible for fruit ripening and for its involvement in a number of plant growth and development processes. Elucidating the mechanisms involved in the ripening of climacteric fruit and the role that ethylene plays in this process have been central to fruit production and the improvement of fruit quality. The biochemistry, genetics and physiology of ripening has been extensively studied in economically important fruit crops and a considerable amount of information is available which ranges from the ethylene biosynthesis pathway to the mechanisms of perception, signaling and control of gene expression. However, there is still much to be discovered about these processes and the objective of this review is to present a brief historic account of how ethylene became the focus of fruit ripening research as well as the development and the state-of- art of these studies at both biochemical and genetic levels.

O etileno, conhecido, sobretudo, como o fitormônio da maturação de frutos, está envolvido em vários processos fisiológicos, atuando na regulação da expressão de grande número de genes. O clone parcial de cDNA ER49 foi previamente isolado mediante a técnica do "Differential Display". A obtenção e a análise da seqüência completa confirmaram as homologias significativas com fatores de elongação da síntese de proteínas do tipo Ts. Tais proteínas são responsáveis pela reciclagem do complexo EF-Tu•GDP, que atua na ligação do aminoacil-RNAt ao ribossomo. A predição da seqüência protéica permitiu a detecção de um peptídio sinal de endereçamento para a mitocôndria e dos domínios TS-N e EF-Ts. A modelagem da estrutura terciária mostrou que ER49 poderia interagir com EF-Tu•GDP, para promover a reciclagem de GDP em GTP. Estudos de expressão durante a maturação do fruto de tomate (Lycopersicon esculentum Mill.), var. Micro-Tom, foram realizados por RT-PCR, revelando que ER49 tem sua expressão induzida nos últimos estádios de maturação do fruto. Os resultados da análise do DNA genômico por Southern blot sugerem que ER49 faz parte de uma família multigênica de 2-3 elementos. Através de ER49, gene mitocondrial regulado pelo etileno, surgem novas perspectivas para o estudo da regulação de genes em nível pós-transcricional e de sua relação com o metabolismo respiratório.

The plant hormone ethylene, known as the fruit ripening hormone, plays a role in several physiological processes and is involved in the regulation of gene expression. The partial cDNA clone ER49 was previously isolated by Differential Display screening. Full-length sequence analysis has confirmed significant homologies with TS protein synthesis elongation factors. These proteins are responsible for GDP exchange in the EF-Tu•GDP complex which is necessary for aminoacyl-RNAt binding to ribosome. Peptidic sequence prediction allowed the detection of a mitochondrial targeting signal and TS-N and EF-Ts domains. The modeling of tertiary protein structure has shown that ER49 could interact with EF-Tu•GDP and promote GTP exchange. Expression patterns during tomato fruit ripening (Lycopersicon esculentum Mill.), var. Micro-Tom were obtained by RT-PCR, showing that ER49 is up-regulated in the last stages of fruit ripening. Southern analysis suggested that ER49 is encoded by a multigene family of 2-3 elements. Study of this gene has opened new perspective on the understanding of post transcriptional gene regulation by ethylene and its relationship with the respiratory metabolism.

Objetivou-se estudar o comportamento fisiológico de frutos de tomateiro (Lycopersicon esculentum, Mill.), da cv. Kadá, transformados geneticamente, via Agrobacterium tumefaciens, com o clone de DNA pMEL1, em orientação antisenso, e de frutos desta mesma cultivar, não transformados. O estudo fisiológico foi realizado avaliando-se a duração do ciclo de maturação dos frutos, amadurecidos na planta e após a colheita no estádio verde-maduro, e sua produção de etileno. Os frutos transformados amadurecidos na planta tiveram um ciclo total médio de 27 dias, enquanto os amadurecidos após a colheita, tiveram este intervalo prolongado a 50 dias. Ao contrário, os tomates não transformados apresentaram um ciclo de maturação mais acelerado quando colhidos no estádio verde-maduro, em relação aos frutos amadurecidos nas plantas. Os valores foram, em média, de 20 e 30 dias, respectivamente. Estes resultados estão correlacionados com as variações na produção de etileno observada nos dois genótipos. Frutos não transformados produziram, em média, 10,46 nL de etileno.g-1.h-1, enquanto os transgênicos tiveram sua produção de etileno diminuída para 0,13 nL.g-1.h-1. Pode-se concluir, então, que a redução da produção de etileno, verificada nos tomates transformados, é necessária, mas não é suficiente para prolongar o ciclo de maturação e aumentar a durabilidade dos frutos. Para que isto ocorra, é necessário que se proceda à colheita dos tomates no estádio verde-maduro.

This work was carried out to study the physiological behaviour of tomato fruits (Lycopersicon esculentum, Mill.), var. Kada, geneticaly transformed, via Agrobacterium tumefaciens, with the pMEL1 DNA clone, in antisense orientation, and nontransformed ones, from the same variety, growth in a greenhouse. The physiological study was conducted for evaluating the ripening cycle of fruits, ripened attached and detached from the plants in the mature-green stage , and their ethylene production. On average, tranformed tomatoes that ripened atached to the plants had a ripenig cycle of 27 days, whereas in detached fruits, this time was 50 days. Instead of it, nontransformed tomatoes showed a faster ripening process when ripened detached to the plants (20 days), whereas in attached fruits this period was 30 days, on average. These results were related to ethylene production. Tomato fruits from nontransformed plants produced 13,46 nL ethylene.g-1.h-1, and the transformed fruits showed an ethylene production almost 100 times smaller than nontransformed ones ( 0,13 nL ethylene.g-1.h-1). Thus, the conclusion reached is that the reduction of ethylene production observed in transgenic tomatoes is necessary but it is not enough to extend the ripening cycle and to increase the period of time that the fruits will last. To achieve this, it is necessary to harvest the fruits in the mature-green stage.

Com o objetivo de caracterizar, por via imunoquímica, a enzima ACC (ácido 1-carboxílico-1-aminociclopropano) oxidase em frutos climatéricos, foram preparados anticorpos policlonais específicos para esta proteína. Utilizou-se, como antígeno, uma proteína recombinante, produzida em Escherichia coli K38/pGP1,2, contendo o vetor de expressão pT7-7A4 no qual foi inserido um clone de DNA da ACC oxidase. A especificidade dos anticorpos foi demonstrada pela técnica de "Western blot", a partir de extratos protéicos de maçãs e tomates em diferentes estágios de maturação. Verificou-se que o aumento da produção de etileno, quando os frutos passaram do estágio pré-climatérico para o climatérico, está diretamente correlacionada com o aumento da síntese da ACC oxidase. Em estágios de maturação mais avançados houve uma redução da produção de etileno e da atividade ACC oxidase, mas esta proteína continuava presente. Quando o "Western blot" foi realizado com tomates transgênicos, onde a produção de etileno e a síntese da ACC oxidase foram inibidos em mais de 95%, nenhuma reação imunoquímica foi detectada. O conjunto de resultados obtidos indica que os anticorpos detectam especificamente ACC oxidase.

Polyclonal antibodies were prepared to characterize the enzyme ACC (1-aminocyclopropane-1-carboxilic acid) oxidase from climateric fruits. The antigen was a recombinant protein obtained from an Escherichia coli K38/pGP1,2, which contained the expression vector pT7-7A4 with one ACC oxidase DNA clone inserted. Antibody specificity was demonstrated by the Western blot technique with protein extracts from apples and tomatoes in different maturation stages. It was observed that the increase in ethylene production which happened when the fruits changed from pre-climateric to climateric stage is directly correlated with an increase in ACC oxidase syntesis. In more advanced maturation stages there was a reduction in ethylene production and ACC oxidase activity, however the protein was still present. There was no immunochemical reaction in Western blot performed with transgenic tomatoes, which had a 95% reduction of etylene production and ACC oxidase syntesis. This results suggest that the antibodies detects ACC oxidase specifically.