Plants have developed physiological and molecular mechanisms to support and adapt to adverse environments. One response to abiotic stress is the accumulation of free proline (PRO). PRO can induce the expression of many genes, which have the proline-responsive element (PRE) in their promoters, nevertheless due to the complexity of interactions between stress factors and various molecular, biochemical and physiological phenomena it is still unclear whether a more efficient PRO accumulation can be considered a biomarker of tolerance in plants. In the present work, we evaluated the accumulation of PRO in two genotypes of sorghum with contrasting tolerance to cold stress. To explore the cause behind the accumulation of proline under cold stress conditions, we identified the Transcription Factors Binding Sites (TFBS) present in the promoter regions in the genes involved in the biosynthesis and degradation of proline in sorghum and other important crops, finding that the untranslated 3 'region P5CS gene contains different TFBS. We found TFBS that could allow the activation of genes involved in proline biosynthesis through the ornithine pathway under cold stress conditions, suggesting that ornithine route can be activated under cold stress conditions. Keywords: Biomarker, Cold, Proline, Sorghum, TFBS. Mexican Journal of Biotechnology 2018, 3(3):77-86 RESUMENLas plantas han desarrollado mecanismos fisiológicos y moleculares para soportar y adaptarse a entornos adversos. Una de las respuestas a condiciones de estrés abiótico es la acumulación de prolina (PRO). PRO puede inducir la expresión de distintos genes, que tienen el elemento de respuesta a prolina (PRE) en sus promotores, sin embargo, debido a la complejidad de las interacciones entre los factores de estrés y varios fenómenos moleculares, bioquímicos y fisiológicos aún no está claro si la acumulación de PRO puede considerarse un biomarcador de tolerancia en las plantas. En el presente trabajo evaluamos la acumulación de PRO en dos genotipos de sorgo con tolerancia contrastante a estrés por frío. Para explorar la causa detrás de la acumulación de prolina ante condiciones de estrés por frío, identificamos los TFBS presentes en las regiones promotoras en los genes involucrados en la biosíntesis y degradación de prolina en sorgo y otros cultivos importantes encontrando que la región 3' no transcrita del gen P5CS contiene diferentes TFBS. Encontramos TFBS que pudieran permitir la activación de los genes involucrados en la biosíntesis de prolina a través de la vía de ornitina ante condiciones de estrés por frío lo que sugiere que dicha vía puede activarse ante condiciones de estrés por frío.

Studies of gene expression are very important for the identification of genes that participate in different biological processes. Currently, reverse transcription quantitative real-time PCR (RT-qPCR) is a high-throughput, sensitive and widely used method for gene expression analysis. Nevertheless, RT-qPCR requires precise normalisation of data to avoid the misinterpretation of experimental data. In this sense, the selection of reference genes is critical for gene expression analysis. At this time, several studies focus on the selection of reference genes in several species. However, the identification and validation of reference genes for the normalisation of RT-qPCR have not been described in amaranth. A set of seven housekeeping genes were analysed using RT-qPCR, to determine the most stable reference genes in amaranth for normalisation of gene expression analysis. Transcript stability and gene expression level of candidate reference genes were analysed in different tissues, at different developmental stages and under different types of stress. The data were compared using the geNorm, NormFinder and Bestkeeper statistical methods. The reference genes optimum for normalisation of data varied with respect to treatment. The results indicate that AhyMDH, AhyGAPDH, AhyEF-1α and AhyACT would be optimum for accurate normalisation of experimental data, when all treatment are analysed in the same experiment. This study presents the most stable reference genes for normalisation of gene expression analysis in amaranth, which will contribute significantly to future gene studies of this species.

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