Individual and combined effects of the rolA, B, and C genes on anthraquinone production in Rubia cordifolia transformed calli.

It is known that the rolA, rolB, and rolC genes of Agrobacterium rhizogenes T-DNA affect processes of plant development and activate the synthesis of secondary metabolites in transformed plant cells. Although a synergistic activity of the rol genes on root formation is well-documented, little is known about their individual and combined action on secondary metabolism. In the present investigation, we provide evidence indicating that individual rolA, rolB, and rolC genes are capable of increasing biosynthesis of anthraquinones (AQs) in transformed calli of Rubia cordifolia. The stimulatory effect was due to the increased transcription of a key gene of AQ biosynthesis, the isochorismate synthase (ICS) gene. The strongest AQ-stimulating activity was shown for an R. cordifolia culture expressing rolB at high levels, where rolB ensured a 15-fold increase of AQ accumulation compared with the control, non-transformed calli. A tyrosine phosphatase inhibitor abolished the rolB-induced increase of AQ production, thus indicating the involvement of tyrosine (de)phosphorylation in the rolB-mediated AQ stimulation. The rolA- and rolC-expressing cultures produced 2.8- and 4.3-fold higher levels of AQs, respectively, when compared with the control calli. However, the effect of rolA, rolB, and rolC on AQ biosynthesis was not synergistic because rolA and rolC apparently attenuated the stimulatory effect of rolB on AQ biosynthesis. Therefore, the rol-gene-mediated signals that promote root formation and those which activate biosynthesis of secondary metabolites seem to have a point of divergence.

CDPK-driven changes in the intracellular ROS level and plant secondary metabolism.

Heterologous expression of a constitutively active calcium-dependent protein kinase (CDPK) gene was previously shown to increase secondary metabolite production in cultured cells of Rubia cordifolia, but the critical question of how CDPK activates secondary metabolism remains to be answered. In this article, we report that the expression of the Arabidopsis CDPK gene, AtCPK1, in R. cordifolia cells caused moderate and stable elevation of intracellular reactive oxygen species (ROS) levels. In contrast, the non-active, mutated AtCPK1 gene did not cause such an effect. The active AtCPK1 also increased cell size, likely by restricting cell division. These results are consistent with the model in which constitutive expression of AtCPK1 mimics the effects of elicitors, acting on secondary metabolism via the activation of ROS production.

Synthesis of echinamines A and B, the first aminated hydroxynaphthazarins produced by the sea urchin Scaphechinus mirabilis and its analogues.

The first total synthesis of two marine aminated hydroxynaphthazarins, echinamines A (3-amino-7-ethyl-2,5,6,8-tetrahydroxy-1,4-naphthoquinone) and B (2-amino-7-ethyl-3,5,6,8-tetrahydroxy-1,4-naphthoquinone), produced by the sea urchin Scaphechinus mirabilis is described. This was achieved from 1,2,4-triacetoxybenzene (13) through a sequence involving double Fries rearrangement of 13, reduction of 3,5-diacetyl-1,2,4-trihydroxybenzene (14), methylation of 3,5-diethyl-1,2,4-trihydroxybenzene (15), simultaneous double acylation of 3,5-diethyl-1,2,4-trimethoxybenzene (16) with a dichloromaleic anhydride-ethyl radical elimination process, methylation of 6,7-dichloro-3-ethyl-2-hydroxynaphthazarin (17), nucleophilic substitution of a chlorine atom by the methoxy group in 6,7-dichloro-3-ethyl-2-methoxynaphthazarin (18), introduction of an amino group via direct substitution of a chlorine atom in 7-chloro-3-ethyl-2,6-dimethoxy- (11) and 7-chloro-2-ethyl-3,6-dimethoxynaphthazarins (12) by an azido group, and functional group deprotection. The synthesis of amino analogues of spinazarin and spinochrome D is also described.

Echinamines A and B, first aminated hydroxynaphthazarins from the sea urchin Scaphechinus mirabilis.

Two new spinochromes, echinamines A (1) and B (2), were isolated from the sea urchin Scaphechinus mirabilis. Compounds 1 and 2 represent the first examples of natural polyhydroxynaphthazarins with a primary amine group. The structures of 1 and 2 were established by analysis of spectroscopic data and synthesis of their dimethyl ethers.

Caffeic acid metabolites from Eritrichium sericeum cell cultures.

Eritrichium sericeum (Boraginaceae) callus and root cultures were established and analyzed for caffeic acid metabolite (CAM) production. Two substances, (-)-rabdosiin and rosmarinic acid, were identified as main CAMs produced by these cultures. The E. sericeum Er-1 root culture accumulated up to 1.5 % and 4.5 % DW of (-)-rabdosiin and rosmarinic acid, respectively. Rabdosiin in the Lithospermum erythrorhizon callus cultures was produced exclusively as the (+)-enantiomer while in both Eritrichium cultures it occurred as the (-)-enantiomer. The E. sericeum Er-1 culture accumulated 3-fold higher levels of CAMs than the L. erythrorhizon culture. A new compound, named eritrichin, was isolated from the cultured E. sericeum cells. The structure of this compound was established as (2R)-3-(3,4-dihydroxyphenyl)-2-[4-(3,4-dihydroxyphenyl)-6,7-dihydroxy-2-naphthoyloxy]propanoic acid on the basis of spectral data.