Studies on the metabolic pathways leading to betanin in Beta vulgaris L. cell cultures through labelling by tyrosine and dihydroxyphenylalanine

The aim of our work is the study of the metabolic pathways leading to betanin in cell cultures of Beta vulgaris L. by incorporation of tyrosine and dihydroxyphenylalanine (dopa).

Experiments with the entire plant. Incorporation trials with ¹⁴C-tyrosine and ¹⁴C-dopa in roots of Beta vulgaris L. led to the identification of a novel biosynthetic intermediate cyclodopa glucoside (CDG) present especially in young roots. We have fully characterized this compound which was found in large amounts in the roots, in conjunction with betanin that is the red-violet dye of red beets. The analysis of the distribution ratio of radioactivity between the two moieties of betanin after hydrolysis show a disproportionate incorporation between the cyclodopa glucoside and the betalamic acid components. Dopamine is identified as an important metabolite in this root.

Experiments with cell cultures. By the initiation of Beta vulgaris L. cell cultures in suspension, with a high betanin content, we were able to proceed with biosynthetic studies. This permitted us to further understand the individual role played by the precursors tyrosine and dopa during the compartimentation of the metabolism when synthesizing CDG and betanin.

In order to perform the biosynthetic studies, standardized cell culture conditions were developed along with optimal incubation conditions using radioactive precursors. An analytical method able to separate all of the metabolites in a single HPLC run, using multiple wavelength detection and continuous measurements of radioactivity was developed as well. The radioactive metabolites that were identified in the cell culture extracts are: tyrosine, dopa, dopamine (the major metabolite), tyramine, CDG and betanin.

The results of a 3 hr incorporation kinetic experiment have confirmed that CDG is a true biosynthetic intermediate with a more rapid incorporation coming from ¹⁴C-tyrosine than from ¹⁴C-dopa. By using a mathematical model to simulate the metabolite's flows between the compartments, our experimental data leads us to propose two metabolically distinct reservoirs for dopa as an explanation of this unbalanced incorporation into CDG and betanin.

For the compartimentation studies, shorter incorporation times (20, 30 and 60 min) have been used, with stationary experimental conditions, in order to guarantee a linear internalisation of precursors over time.

Simultaneous incubation of cultures with ³H-tyrosine and ¹⁴C-dopa using fewer cells shows up a preferential incorporation of tyrosine in CDG with a CDG-³H/ ¹⁴C-ratio larger then the dopa-³H/¹⁴C-ratio.

Competition experiments performed in the presence of the two precursors, given simultaneously with equi molar amounts, alternatively labelled with ¹⁴C and non-radioactive, have demonstrated a preferential ¹⁴C-tyrosine incorporation into CDG and in the dihydroindole-part of betanin. Non-radioactive tyrosine interferes with the internalisation of ¹⁴C-dopa, hence reinforcing our hypothesis of two metabolically distinct dopa compartments: dopa(l) providing a preferential access to CDG and dopa(2) in majority feeding the dopamine and betalamic acid reservoirs.

Experiments made with a dopa-decarboxylase inhibitor, 2-(fluoromethyl)-3-(3,4-dihydroxyphenyl)-alanine (MFMD) confirm that the temporary accumulation of radioactive dopa does not have the same bioavailability for the synthesis of CDG and betalamic acid when generated from ¹⁴C-tyrosine or ¹⁴C-dopa administered to the cells.

A metabolically simplified cellular model is proposed, strengthened by the experimental results showing two metabolically distinct dopa compartments: the dopa(l) compartment is fed by tyrosine and flows directly into CDG and the dopa(2) compartment fed from dopa(l) is the source of betalamic acid and dopamine. CDG and betalamic acid coming from these two different metabolic processes are assembled by condensation to give the betanin that is found in the vacuolar compartment.