Dissecting the sugarcane expressed sequence tag (SUCEST) database: unraveling flower-specific genes

There are almost 260,000 independent clones sequenced from the 5? end in the Sugarcane Expressed Sequence Tag (SUCEST) database, which have been obtained from 37 cDNA libraries prepared from different tissues. This large number of expressed sequence tags (ESTs) provides an opportunity, unprecedented in plants, to perform ?digital differential screening? on selected cDNA libraries. In general, the frequency of a particular EST correlates with transcript accumulation in the tissues from which the cDNA libraries were constructed, so it is possible to compare the whole transcriptome from different tissues using computer-assisted analysis of an EST database. In our research we analyzed sugarcane ESTs according to tissue expression and identified more than 1,000 putative flower-specific genes. The fact that using this technique we were able to identify sugarcane homologues of several genes previously described as pollen-specific justifies this method of assessing tissue specificity. In addition, ESTs similar to genes specific to reproductive organs were detected e.g. a sugarcane gene encoding a meiotic protein essential for assembly of the synaptonemal complex and normal synapsis. This approach also allowed the identification of many flower-specific anonymous sequences that are good candidates for being novel genes involved in plant reproduction. This paper describes the analysis of the gene expression levels of 24 EST clusters during flower development using a ?digital northern blot? constructed from direct EST counts made on the non-normalized sugarcane cDNA libraries.Existem quase 260.000 clones independentes, seqüenciados a partir da extremidade 5?, no banco de dados do SUCEST (Sugarcane Expressed Sequence Tag), os quais foram obtidos a partir de 37 bibliotecas de cDNA preparadas de diferentes tecidos. Este grande número de etiquetas de sequências expressas (ESTs) fornece uma oportunidade, sem precedentes em plantas, de realizar um ?digital differential screening? em bibliotecas de cDNA selecionadas. Geralmente, a frequência de um determinado EST está correlacionada ao acúmulo de transcritos nos tecidos dos quais as bibliotecas de cDNA foram construídas, e desta forma, é possível comparar o transcriptoma completo de diferentes tecidos, usando uma análise computacional de um banco de dados de ESTs. Em nossa pesquisa, analisamos os ESTs de cana-de-açúcar de acordo com sua expressão tecidual e identificamos mais de 1.000 putativos genes específicos de flor. O fato de que usando esta técnica fomos capazes de identificar homológos em cana-de-açúcar, de vários genes previamente descritos como específicos de pólen, sustenta este método de estimar especificidade tecidual. Além disto, ESTs com similaridade a genes específicos de órgãos reprodutivos foram revelados, como por exemplo, o gene que codifica uma proteína meiótica essencial para a montagem do complexo sinaptonêmico e sinapse normal. Esta abordagem também permitiu a identificação de muitas sequências anônimas, específicas de flor, que são boas candidatas para novos genes envolvidos com a reprodução de plantas. Este trabalho descreve a análise dos níveis de expressão gênica de 24 clusters de ESTs, durante o desenvolvimento floral, usando um ?northern blot digital? construído a partir da contagem direta dos ESTs das bibliotecas não-normalizadas de cDNAs de cana-de-açúcar.7784Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

SCI1 Is a Direct Target of AGAMOUS and WUSCHEL and Is Specifically Expressed in the Floral Meristematic Cells

The specified floral meristem will develop a pre-established number of floral organs and, thus, terminate the floral meristematic cells. The floral meristematic pool of cells is controlled, among some others, by WUSCHEL (WUS) and AGAMOUS (AG) transcription factors (TFs). Here, we demonstrate that the SCI1 (Stigma/style cell-cycle inhibitor 1) gene, a cell proliferation regulator, starts to be expressed since the floral meristem specification of Nicotiana tabacum and is expressed in all floral meristematic cells. Its expression is higher in the floral meristem and the organs being specified, and then it decreases from outside to inside whorls when the organs are differentiating. SCI1 is co-expressed with N. tabacum WUSCHEL (NtWUS) in the floral meristem and the whorl primordia at very early developmental stages. Later in development, SCI1 is co-expressed with NAG1 (N. tabacum AG) in the floral meristem and specialized tissues of the pistil. In silico analyses identified cis-regulatory elements for these TFs in the SCI1 genomic sequence. Yeast one-hybrid and electrophoresis mobility shift assay demonstrated that both TFs interact with the SCI1 promoter sequence. Additionally, the luciferase activity assay showed that NAG1 clearly activates SCI1 expression, while NtWUS could not do so. Taken together, our results suggest that during floral development, the spatiotemporal regulation of SCI1 by NtWUS and NAG1 may result in the maintenance or termination of proliferative cells in the floral meristem, respectively

A tobacco flower-specific gene encodes a polyphenol oxidase

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Sci1 Is A Component Of The Auxin-dependent Control Of Cell Proliferation In Arabidopsis Upper Pistil

To characterize the recently described SCI1 (stigma/style cell cycle inhibitor 1) gene relationship with the auxin pathway, we have taken the advantage of the Arabidopsis model system and its available tools. At first, we have analyzed the At1g79200 T-DNA insertion mutants and constructed various transgenic plants. The loss- and gain-of-function plants displayed cell number alterations in upper pistils that were controlled by the amino-terminal domain of the protein. These data also confirmed that this locus holds the functional homolog (. AtSCI1) of the Nicotiana tabacum SCI1 gene. Then, we have provided some evidences the auxin synthesis/signaling pathways are required for downstream proper AtSCI1 control of cell number: (a) its expression is downregulated in yuc2yuc6 and npy1 auxin-deficient mutants, (b) triple (. yuc2yuc6sci1) and double (. npy1sci1) mutants mimicked the auxin-deficient phenotypes, with no synergistic interactions, and (c) the increased upper pistil phenotype in these last mutants, which is a consequence of an increased cell number, was able to be complemented by AtSCI1 overexpression. 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Determinação da viabilidade de protoplastos irradiados de laranja 'pêra' Determination of viability of irradiated 'pera' orange protoplasts

Estudou-se a viabilidade de protoplastos de laranja 'Pêra' (Citrus sinensis Osbeck), submetidos a diferentes doses de radiação gama, com a finalidade de determinar a dose letal (DL) 50 - dose que causa 50% de letalidade. Empregou-se a análise por fluorescência, utilizando-se o corante diacetato de fluoresceína (DAF): suas diluições testadas - 1:50; 1:100 e 1:150 - não mostraram diferenças significativas entre si, tendo sido possível o uso da maior diluição para a determinação da viabilidade dos protoplastos. A viabilidade mostrou-se inversamente proporcional às doses de radiação gama e a DL 50 foi cerca de 41 Gy. Os protoplastos não irradiados apresentaram até 84% de viabilidade, quando esta foi estudada logo após o isolamento daqueles.<br>The viability of 'Pera' orange (Citrus sinensis Osbeck) protoplasts, submitted to different dosages of Gamma radiation, was studied to determine the lethal dose (DL) 50. The analysis by fluorescence was employed using Fluorescein diacetate (FDA). The dilutions of FDA (1:50; 1:100 and 1:150) did not show any statistical difference: Then it was possible to use the 1:150 dilution in order to determine the protoplasts viability. The viability was inversaly proportional to Gamma radiation and the DL 50 was about 41 Gy. The non-irradiated protoplasts had their viability up to 84% when tested as soon after their isolation