Study of glutamate addition on secondary metabolism of Rubia tinctorum cell suspension cultures

PERASSOLO M; GIULIETTI, A M; RODRIGUEZ TALOU, J

Barcelona, España

Congreso; 13th European Congress on Biotechnology.16-19 septiembre 2007,; 2007

Anthraquinones (AQs) are a group of secondary metabolites that have been widely used for medicinal purposes. Their chemical structure is derived from isopentenyl diphosphate via the terpenoid pathway and o-succinylbenzoic acid, which is produced from a–ketoglutaric acid and isochorismic acid. The latter is generated from chorismic acid, the end-product of the shikimate pahtway and the branch point for the synthesis of phenylpropanoid and anthraquinones (AQs) in Rubia tinctorum secondary metabolism (1). It has been proposed that proline could affect the pentose phosphate pathway (PPP) since its synthesis from glutamate generates 2 molecules of NADP+, which act as cofactors of the first two enzymes in the PPP, the glucose-6-phosphate dehydrogenase (G6PD) and the 6-phosphogluconate dehydrogenase (2). Stimulating the proline cycle of biosynthesis and degradation could induce the PPP generating an excess of erithrose-4-phosphate, which is the substrate of the shikimate pathway (2). In this work, we assayed the effect of glutamate addition on plant suspension cultures of R. tinctorum in order to study its effect on secondary metabolic pathways.  Suspension cultures of R.tinctorum were treated with 5 mM of glutamate, and determination of anthraquinones, total phenolics and proline were carried out at 1, 2, 3, 4 and 8 days of culture. Enzymatic activities of G6PD and L-phenylalanine ammonia-lyase (PAL), the key enzyme in phenolpropanoids biosynthesis, were also assayed.  Suspension cultures treated with 5 mM glutamate showed higher levels of AQs at 4 (30%) and 8 (24%) days of culture compared to non-treated suspensions. Total phenol content also increased in treated cells at 2 (11%) and 4 (11%) days of culture. Proline accumulation was observed at the beginning of the experiment (1 and 2 days), showing an increase of 55 and 110% in glutamate treated-cultures. Although the proline cycle was stimulated with the addition of glutamate, no induction of G6PD and PAL was observed during the experiment