présentée à la Faculté des Sciences
de l'Université de Lausanne
en vue de l'obtention du grade de docteur ès Sciences
Giampiero Franco TREZZINI
Dipl. Sc. Nat. ETHZ
The betalains are a class of chromo-alkaloid belonging to the general group of compounds known as secondary metabolises. These molecules are divided into two colour groups, the violet betacyanins and the yellow betaxanthins, both of which possess the common tyrosine-derived chromophore, betalamic acid The pigments are found in different tissues (flowers, fruits, roots etc.) of plants from the order Caryophyllales and their presence mutually excludes the appearance of the more commonly found plant pigments, the anthocyanins. On the one hand, the betalains can be considered as interesting markers to the state of cellular differentiation (Girod, 1989) and, on the other, the betaxanthins, alongside the betacyanins, may have a role to play as natural colorants in the food industry. Presented here is a study of betalain genetics in Portulaca grandiflora (large flowered purslane) which was undertaken to determine the number of genes involved the regulation of the biosynthesis of these pigments. A model is proposed, integrating our current knowledge of the genetics and biochemistry in this species, to describe the regulation and spatial orientation (subcellular localisation) of betalain biosynthesis.
In P. grandiflora three genes are believed to be involved in the control of betalain biosynthesis. The first, gene C, is responsible for the formation of the chromophore. It is likely that gene C codes for the enzyme DOPA-4,5-dioxygenase, capable of catalysing the extradiol cleavage of DOPA, the betalain precursor. Gene R is responsible for the production of cyclo-DOPA (glycosylated). The substrates for this reaction are thought to be both tyrosine and/or DOPA. Condensation of betalamic acid with cyclo-DOPA (glycosylated) results in the formation of a betacyanin (lmax approx. 540 nm). Equally, betalamic acid may be conjugated with amino acids or other amines to give betaxanthins (lmax approx. 478 nm). The third gene, I, inhibits or modulates the final condensation reaction. It has been established that R is an incomplete dominant gene. Gene's C and I are both dominant with each affecting the function of the other. R and I exhibit close linkage (5.3 + 0.7 cMorgans) and can exist in at least two allelic forms. Gene C segregates independently from R and I. Evidence was obtained demonstrating the presence of a transposable element, which exhibited an elevated frequency of transposition when plants were cross fertilised vitro .
A method based on HPLC separation was developed for the identification and quantification of betalain pigments in the order Caryophyllales. Spectrophotometric data (molar extinction coefficient and lmax ) are given for pigments described as occurring naturally and for other semi-synthetic analogues generated by conjugation of betalamic acid, derived from either betanin or indicaxanthin, with a range of protein and non-protein amino acids.
Two new yellow pigments were identified in petals of P. grandiflora. These two betaxanthins were the condensation products of betalamic acid with either tyrosine or glycine, for which the trivial names portulacaxanthin II and portulacaxanthin III were coined, respectively. These pigments along with dopaxanthin, miraxanthin V, vulgaxanthin I and betanin constituted the major betalain signals appearing in petals of the different phenotypes studied.
The HPLC technique was also used to determine the kinetics of pigment biosynthesis in flower buds. The accumulation of yellow pigments was biphasic. In the first phase, yellow pigments derived from two molecules of tyrosine (portulacaxanthin II, dopaxanthin and miraxanthin V) were accumulated. The onset of the second phase, 15 hours after the initiation of the first, was characterised by the accumulation of vulgaxanthin I and portulacaxanthin III. Betanin was accumulated during the second phase.
Based on experimental data, the following model for betalain biosynthesis is proposed: Betalamic acid and the glycosylated form of cyclo-DOPA are produced in the cytoplasm where they may condense to form a betacyanin that is subsequently transported into the vacuole for accumulation. The free chromophore may also be transported, under the regulation of gene I, directly into the vacuole where it may become conjugated with amino acids, present in high concentration, resulting in formation of the betaxanthins.
P. grandiflora was also used for the development of an in vitro model system to study cellular differentiation. Cell lines were established from plants both capable and incapable of betalain synthesis in their tissues. These phenotypic characteristics were maintained in cultured tissues. Changes in the concentration of auxin were accompanied by physiological and biochemical changes within the cells.