Bagnaresi et al - Biomolecular approaches for studying potato tuber cold sweetening

Paolo Bagnaresi1*, Anna Moschella2, Bruno Parisi2, Pierdomenico Perata3 e Paolo Ranalli4
1 CRA-GPG-Centro di Genomica e Post-Genomica Animale e Vegetale, via S. Protaso 302, 29017 Fiorenzuola d’Arda (PC)
2 CRA - Centro di Ricerca per le Colture Industriali, via di Corticella 133, 40128 Bologna
3 Plant and Crop Physiology Lab , Scuola Superiore Sant’Anna, p.za Martiri della Libertà, 56127 Pisa
4 CRA - Dipartimento di Trasformazione e Valorizzazione dei Prodotti Agro-industriali, Roma.

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Key words: Maillard reaction, acrylamide, asparagine, microarray.

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Abstract

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As it is known by more than 120 years, cold incubation of tubers cause accumulation of sugars (mainly sucrose, glucose and fructose) at the expenses of starch. This is detrimental for tuber quality, as, upon cooking at high temperatures, an excess of dark, bitter tasting melanoidins are produced due to the Maillard reaction involving reducing sugars and free amino acids. In recent years, concern for phenomenon has further risen since a specific type of Maillard reaction involving the amino-acid asparagine (abundant in potato tubers) and reducing sugars has been shown to produce the neurotoxic and genotoxic compound acrylamide. This unexpected finding was prompted by investigations on the accidental release of acrylamide in the environment. Attempts have been conducted in order to identify varieties with reduced asparagine level, but the extent of reducing sugar accumulation appears by far the major parameter affecting acrylamide production in potato derivatives and thus brings the focus back to the sweetening phenomenon. Research on potato cold-induced sweetening (CIS) has implicated several carbohydrate-associated genes in the process. However, still many uncertainties exist, as the relative contribution of each gene to the process is often unclear, possibly as the consequence of the heterogeneity of experimental systems. Some enzymes associated to CIS, as ß-amylases and invertases, still await identification at the sequence level. Additionally, little is known about early events triggering CIS and involvement/association to CIS of genes other than CAG. Many of those uncertainties could be resolved by profiling experiments, but no GeneChip is available for potato and the production of the potato cDNA spotted array (TIGR) has recently been discontinued. In order to obtain an overall picture of early transcriptional events associated to CIS, we investigated whether the commercially-available tomato Affymetrix GeneChip could be used to discern among potato cold-responsive gene family members to be further studied in detail by Real-Time (RT)-PCR (qPCR). A tomato-potato global match file (GMF) was generated by matching tomato targets to potato ESTs in order to establish of a core set of highly homologous genes. A further, low-scale probe-level (oligonucleotide) approach was also explored to maximize reliability of the heterologous dataset for genes of particular interest. Several cold-responsive genes were identified, and their expression pattern was studied in detail by qPCR over a 26-d time course. We detected biphasic behaviour of mRNA accumulation for CAG and our combined GeneChip-qPCR data identify, at the sequence level, enzymatic activities as ß- amylases and invertases previously reported to take part to CIS. Scrutiny of validated GeneChip data further unveils important processes accompanying CIS, such as the induction of redox- and hormone-associated genes. This strategy revealed essential for accurate heterologous dataset interpretation and suggests that similar approaches can fruitfully be conducted for other species. Transcript profiling of early events associated to CIS discloses a complex network of events involving sugars, redox and hormone signalling which may be either serially linked or act in parallel. Identification at the sequence level of various enzymes long known to take part to CIS provides molecular tools for further understanding of the phenomenon.

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