Echeverry Solarte, Morgan

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    Quantification of the transcriptional activity of genes associated with cyanogenesis in Cassava (Manihot esculenta Crantz)
    (2008) Echeverry Solarte, Morgan; Siritunga, Dimuth; College of Arts and Sciences - Sciences; Macchiavelli, Raúl E.; Rodríguez Minguela, Carlos M.; Ríos Velázquez, Carlos; Department of Biology; Porch, Timothy
    Cassava has cyanogenic glycosides in its roots and leaves. These compounds function in plants as protective agents against herbivores as well as the translocable form of reduced nitrogen. The genes involved in the cyanogenic pathway [CYP79D1, CYP79D2, linamarase, α-hydroxynitrile lyase (HNL) and β-cyanoalanine synthase (β-CAS)] have been identified, though very little is known regarding their transcriptional regulation. Our work aimed to identify the differential expression of these genes using conventional PCR and Real Time PCR in cassava plants of different cultivars grown in vitro under different environment conditions. Four experiments were designed to accomplish this objective: 1. Semiquantitative comparison of the transcriptional activity in leaves and roots of two cassava cultivars using conventional PCR; 2. Quantitative comparison of the transcriptional activity in leaves and roots of three cassava cultivars using Real-Time PCR; 3. Quantitative comparison of the transcriptional activity in leaves and roots of two cassava cultivars under reduced nitrogen stress using Real-Time PCR; 4. Quantitative comparison of the transcriptional activity in leaves and roots of two cassava cultivars under temperature stress using Real-Time PCR. Statistical analyses demonstrated a higher expression of the genes in leaves relative to the roots, consistent with the fact that cyanogens are synthesized predominantly in the leaves and subsequently transported to the roots where they are converted to proteins. In the comparison of the expression among three cultivars, the results suggest that differences in cyanide content in roots could be explained by the coordinated cyanogenic glycoside synthesis in leaves and the linamarase catabolic step in the roots. The comparison under reduced nitrogen stress shows the impact of this environmental condition over the expression pattern of these genes. Thus, it is reported that CYP79D1/D2 genes increase their transcriptional activity in inverse relation to reduced nitrogen concentration in roots after one day of stress; while the β-CAS gene responds in direct relation to reduced nitrogen concentrations in leaves and roots primarily after 10 days of stress. The results obtained from the temperature stress experiment illustrate an influence of temperature on β-CAS gene expression, though this result was not considered a direct effect of temperature stress over the general cyanogenic glycoside pathway in cassava. Finally, multivariate analysis of the expression of those genes suggest a correlation between the expression of linamarse and HNL genes (involved in the breakdown of linamarin) and among CYP79D1/D2 and β-CAS. This fact is corroborated by the responses of CYP79D1/D2 and β-CAS genes under reduced nitrogen stress, as well as the antecedents that illustrate the linamarase and HNL protein are located separately in the cell wall. This is the first study that evaluate the transcriptional activity of the genes (known to date) involved in the cyanogenic glycoside metabolism in an integral way. The results show the complexity of cyanogenic glycosides metabolism in cassava.