RESUMEN
BACKGROUND: MicroRNAs (miRNAs) are small regulatory RNAs, some of which are conserved in diverse plant genomes. Therefore, computational identification and further experimental validation of miRNAs from non-model organisms is both feasible and instrumental for addressing miRNA-based gene regulation and evolution. Sugarcane (Saccharum spp.) is an important biofuel crop with publicly available expressed sequence tag and genomic survey sequence databases, but little is known about miRNAs and their targets in this highly polyploid species. RESULTS: In this study, we have computationally identified 19 distinct sugarcane miRNA precursors, of which several are highly similar with their sorghum homologs at both nucleotide and secondary structure levels. The accumulation pattern of mature miRNAs varies in organs/tissues from the commercial sugarcane hybrid as well as in its corresponding founder species S. officinarum and S. spontaneum. Using sugarcane MIR827 as a query, we found a novel MIR827 precursor in the sorghum genome. Based on our computational tool, a total of 46 potential targets were identified for the 19 sugarcane miRNAs. Several targets for highly conserved miRNAs are transcription factors that play important roles in plant development. Conversely, target genes of lineage-specific miRNAs seem to play roles in diverse physiological processes, such as SsCBP1. SsCBP1 was experimentally confirmed to be a target for the monocot-specific miR528. Our findings support the notion that the regulation of SsCBP1 by miR528 is shared at least within graminaceous monocots, and this miRNA-based post-transcriptional regulation evolved exclusively within the monocots lineage after the divergence from eudicots. CONCLUSIONS: Using publicly available nucleotide databases, 19 sugarcane miRNA precursors and one new sorghum miRNA precursor were identified and classified into 14 families. Comparative analyses between sugarcane and sorghum suggest that these two species retain homologous miRNAs and targets in their genomes. Such conservation may help to clarify specific aspects of miRNA regulation and evolution in the polyploid sugarcane. Finally, our dataset provides a framework for future studies on sugarcane RNAi-dependent regulatory mechanisms.
Asunto(s)
MicroARNs/genética , Proteínas de Plantas/genética , ARN de Planta/genética , Saccharum/genética , Secuencia de Bases , Sitios de Unión/genética , Biocombustibles , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , MicroARNs/química , MicroARNs/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Filogenia , Proteínas de Plantas/clasificación , Precursores del ARN/química , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN de Planta/química , ARN de Planta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharum/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
BACKGROUND: Sucrose content is a highly desirable trait in sugarcane as the worldwide demand for cost-effective biofuels surges. Sugarcane cultivars differ in their capacity to accumulate sucrose and breeding programs routinely perform crosses to identify genotypes able to produce more sucrose. Sucrose content in the mature internodes reach around 20% of the culms dry weight. Genotypes in the populations reflect their genetic program and may display contrasting growth, development, and physiology, all of which affect carbohydrate metabolism. Few studies have profiled gene expression related to sugarcane's sugar content. The identification of signal transduction components and transcription factors that might regulate sugar accumulation is highly desirable if we are to improve this characteristic of sugarcane plants. RESULTS: We have evaluated thirty genotypes that have different Brix (sugar) levels and identified genes differentially expressed in internodes using cDNA microarrays. These genes were compared to existing gene expression data for sugarcane plants subjected to diverse stress and hormone treatments. The comparisons revealed a strong overlap between the drought and sucrose-content datasets and a limited overlap with ABA signaling. Genes associated with sucrose content were extensively validated by qRT-PCR, which highlighted several protein kinases and transcription factors that are likely to be regulators of sucrose accumulation. The data also indicate that aquaporins, as well as lignin biosynthesis and cell wall metabolism genes, are strongly related to sucrose accumulation. Moreover, sucrose-associated genes were shown to be directly responsive to short term sucrose stimuli, confirming their role in sugar-related pathways. CONCLUSION: Gene expression analysis of sugarcane populations contrasting for sucrose content indicated a possible overlap with drought and cell wall metabolism processes and suggested signaling and transcriptional regulators to be used as molecular markers in breeding programs. Transgenic research is necessary to further clarify the role of the genes and define targets useful for sugarcane improvement programs based on transgenic plants.