Functional and comparative analysis of THI1 gene in grasses with a focus on sugarcane.

De novo synthesis of thiamine (vitamin B1) in plants depends on the action of thiamine thiazole synthase, which synthesizes the thiazole ring, and is encoded by the THI1 gene. Here, we investigated the evolution and diversity of THI1 in Poaceae, where C4 and C3 photosynthetic plants co-evolved. An ancestral duplication of THI1 is observed in Panicoideae that remains in many modern monocots, including sugarcane. In addition to the two sugarcane copies (ScTHI1-1 and ScTHI1-2), we identified ScTHI1-2 alleles showing differences in their sequence, indicating divergence between ScTHI1-2a and ScTHI1-2b. Such variations are observed only in the Saccharum complex, corroborating the phylogeny. At least five THI1 genomic environments were found in Poaceae, two in sugarcane, M. sinensis, and S. bicolor. The THI1 promoter in Poaceae is highly conserved at 300 bp upstream of the start codon ATG and has cis-regulatory elements that putatively bind to transcription factors associated with development, growth, development and biological rhythms. An experiment set to compare gene expression levels in different tissues across the sugarcane R570 life cycle showed that ScTHI1-1 was expressed mainly in leaves regardless of age. Furthermore, ScTHI1 displayed relatively high expression levels in meristem and culm, which varied with the plant age. Finally, yeast complementation studies with THI4-defective strain demonstrate that only ScTHI1-1 and ScTHI1-2b isoforms can partially restore thiamine auxotrophy, albeit at a low frequency. Taken together, the present work supports the existence of multiple origins of THI1 harboring genomic regions in Poaceae with predicted functional redundancy. In addition, it questions the contribution of the levels of the thiazole ring in C4 photosynthetic plant tissues or potentially the relevance of the THI1 protein activity.

Transcriptional Regulation of zma-MIR528a by Action of Nitrate and Auxin in Maize.

In recent years, miR528, a monocot-specific miRNA, has been assigned multifaceted roles during development and stress response in several plant species. However, the transcription regulation and the molecular mechanisms controlling MIR528 expression in maize are still poorly explored. Here we analyzed the zma-MIR528a promoter region and found conserved transcription factor binding sites related to diverse signaling pathways, including the nitrate (TGA1/4) and auxin (AuxRE) response networks. Accumulation of both pre-miR528a and mature miR528 was up-regulated by exogenous nitrate and auxin treatments during imbibition, germination, and maize seedling establishment. Functional promoter analyses demonstrated that TGA1/4 and AuxRE sites are required for transcriptional induction by both stimuli. Overall, our findings of the nitrogen- and auxin-induced zma-MIR528a expression through cis-regulatory elements in its promoter contribute to the knowledge of miR528 regulome.

Regulação do Proto-oncogene c-myc pelo Fator de Transcrição NFAT1

A família de fatores de transcrição NFAT (Fator Nuclear de Células T ativadas) tem diferentes funções regulatórias no ciclo celular, apoptose, diferenciação celular e angiogênese. O proto-oncogene c-myc, que está envolvido em todos estes mecanismos, é reprimido em alguns modelos, em resposta à Ciclosporina A, que inibe a ativação das proteínas NFAT. Dados anteriores de nosso laboratório mostraram que os linfócitos de camundongos NFAT1-/- sensibilizados com ovalbumina, apresentaram níveis aumentados de c-MYC quando comparados com os linfócitos dos camundongos NFAT1+/+. Desta forma, o objetivo deste trabalho foi avaliar se o NFAT1 regula diretamente a expressão de c-MYC. Este estudo mostrou que linfócitos T de camundongos NFAT1-/- naives superexpressam c-MYC em relação aos linfócitos NFAT1+/+, através de ensaios de PCR em tempo real. Uma análise de bioinformática encontrou sete supostos sítios de ligação para NFAT no promotor de c-myc, conservados em humanos e camundongos. Três desses sítios foram confirmados por um ensaio de mudança de mobilidade eletroforética, incluindo o sítio proximal, que é regulado por NFAT2. Além disso, um ensaio de imunoprecipitação de cromatina com linfócitos murinos demonstrou que o NFAT1 se liga diretamente ao promotor de c-myc in vivo. Estes resultados sugerem que o NFAT1 tem um importante papel na regulação do promotor de c-myc, aparentemente regulando negativamente sua expressão.

The Nuclear Factor of Activated T Cells (NFAT) family of transcription factors has different regulatory functions in the cell cycle, apoptosis, cell differentiation and angiogenesis. The c-myc proto-oncogene, which is also involved in those mechanisms, is repressed, in some models, in response to Cyclosporine A that inhibits NFAT activation. Previous data of our laboratory found that lymphocytes from NFAT1-/- mice sensitized with ovalbumin presented higher levels of c-MYC mRNA when compared with the NFAT1+/+. Hence, the aim of this work was to evaluate whether the NFAT1 directly regulates the c-MYC expression. This study showed that T lymphocytes from NFAT1-/- naive mice overexpress c-MYC mRNA when compared with the NFAT1+/+ mice assessed by Real Time PCR. A bioinformatic analysis found seven putative NFAT binding sites in the c-myc promoter, conserved in human and mouse. Three of them were confirmed by an Eletrophoretic Mobility Shift Assay, including the proximal site, which is upregulated by NFAT2. Additionally, a Chromatin Immunoprecipitation Assay with mouse T lymphocytes demonstrated that NFAT1 directly binds to c-myc promoter in vivo. These findings suggest that NFAT1 plays an important role in the regulation of c-myc promoter apparently through the inhibition of this expression.