Psoralea Species

Morphology and Distribution

The genus Psoralea, L. (family Leguminosae) contains approximately 120 species distributed on the five continents. These plants are mainly found in South Africa (45 species), North America (30 species), and Australia (15 species). However, Psoralea plants are also present in Asia, South America, North Africa, and the mediterranean region. They are annual or perennial and they usually grow in arid or semi-arid zones. For instance, Australian Psoraleae are distributed in large parts of the bush when Eucalyptus and Acacia are represented. The unique European species, Psoralea bituminosa, is a perennial plant from the mediterranean region and therefore is also able to resist drought and hot climate. The plants establish a symbiosis with Rhizobium bacteria of the “cowpea” type (slow-growing Rhizobium now classed as Brady rhizobium). Therefore, the plant growth is strongly dependent on the root nodulation which occurs with these bacteria. The morphology, of the genus Psoralea is very heterogeneous. The general shape can be herbaceous, as in P. cinerea or P. plumosa, or bushy as in P. martinii (all of them originating from Australia). Most species have leaves composed of three unequal leaflets, but some of them have one-foliated leaves, like P. martinii or the Indian P. corylifolia. The flowers have the general shape of Papilionoideae underfamily, and are usually purple or blue. The fruit is a small pod (3 to 8 mm long), indehiscent, one-seeded, and without albumen.

Most Psoralea plants have no use for humans, but are of some interest in agriculture. They are known to improve the value of pastures in hot regions. Gutteridge and Whiteman (1975) studied the possible use of local Psoralea species as forage. They are able to resist arid and semi-arid climates, which is not the case for the major part of the grasses and legumes that were introduced from the Old World. However, a major problem was to obtain seeds enough to realize larger-scale experiments or field trials.

Some of the North American Psoralea, like P. canescens or P. esculenta, possess a thick bulbous root. These bulbs were cooked and eaten by the Indians or the first colonists and the French name pommes de prairies was given to them.

Summary and Conclusions

Callus lines from P. cinerea or hairy root lines from various Psoralea species did not produce detectable levels of psoralen and angelicin, the two furanocoumarins found in this genus. Until now, other plants containing furanocoumarins were shown to still have the possibility to synthesize these metabolites with in vitro culture systems. Further experiments were conducted with selected callus strains. The basal Z medium was modified with glucose instead of sucrose, CuS04, and chitosan as for the transformed roots, and also a 2,4-D starvation experiment. Whatever the treatment, no furanocoumarins could be detected, showing that the calli were strongly recalcitrant to synthesize these products.

The failure of P. cinerea calli to produce a secondary metabolite well represented in the whole plant is not an exception at all in in vitro cultures. This phenomenon is generally attributed to the high degree of differentiation and sophistication, reached only by the whole plant, which is required to obtain the synthesis. In the case of Psoralea plants, histological or cellular localization of furanocoumarins has been little studied, but specific accumulating cells were already found in P. bituminosa embryo. This evidence could support the previous hypothesis of highly differentiated cells which could synthesize or store the metabolites. Our own studies performed with various Psoralea species and organs have demonstrated that the furanoucoumarin content can vary greatly within a short time period. These results could suggest a possible translocation of the molecules in the plant which is impossible within the callus cultures. This could explain the lack of furanocoumarins in the 59 P. cinerea callus lines that were analyzed.

In the case of transformed roots, experiments conducted on macroelements with Lachl strain demonstrated clearly that the standard B5 medium is close to optimal for the growth of hairy roots. Other medium modification and elicitor experiments were performed on Lach5, Lach6, and Cin3: chitosan, glucose, and CuS04 had a drastic effect on growth limitation. Differences in the effect of sucrose and glucose upon culture growth have already been reported as well as a pronounced limitation of growth by glucose. The effect of chitosan can be attributed to a permeabilization of the cells, as already mentioned. CuS04 also tends to limit the growth, although it is somewhat less pronounced than with glucose or chitosan. This is undoubtedly related to a copper toxicity in the cultures.

Concerning furanocoumarins in transformed roots, a glucosylated precursor was probably found, at a low concentration. Therefore, hairy roots seem to have the capacity to synthesize furanocoumarin structures very close to the final molecules (psoralen or angelicin). It is possible that the two final enzymes leading to psoralen could be missing in the roots, which could explain the lack of furanocoumarins in transformed root cultures. From the three hairy root samples that were submitted to a β-glucosidase hydrolysis, Cin3 cultivated with glucose (in the dark) led to the same recovery of the marmesin precursor as Lach5 (control under the light) or Lach6 (with CuS04 in the dark). Glucose failed to activate the glucosylation reaction of this secondary metabolite as hypothetized by Cresswell et al. (1989).

Although Psoralea in vitro cultures did not synthesize psoralen or angelicin, they produced coumestrol and its precursor daidzein. The HPLC chromato-grams obtained with a P. cinerea leaf sample and a callus line or transformed root allow some hypotheses. It is clear that psoralen and angelicin are predominantly produced in the whole plant to the detriment of coumestrol and daidzein. The exact opposite happens in the case of in vitro cultures. Furanocoumarins and isoflavones derive from the same branch point of phenylpropanoid metabolism (): 4-coumarate. In the case of furanocoumarins, 4-coumarate is 1-hydroxylated by an ortho-hydroxylase whereas 4-coumarate is taken up by Co A ligase to give 4-coumaroyl-CoA with isoflavones. It can be then hypothetized that ortho-hydroxylase activity decreases to the benefit of 4-coumarate Co A ligase in callus and hairy root cultures. Besides, this lack of ortho -hydroxylase activity can be related to the proposed feedback inhibition of furanocoumarin synthesis. Because translocation is impossible within in vitro cultures, the marmesin precursor could accumulate, and repress the ortho -hydroxylase synthesis even at low concentration.

The medium modification and elicitor experiments that were carried out with transformed roots led to a quite surprising result. Compared with control B5 medium, all the treatments gave practically no extra content of coumestrol and daidzein in the dry matter. On the contrary, quantifications performed in the liquid medium revealed that the chitosan treatment especially produced far more coumestrol and diadzein than the controls. Glucose, although limiting the growth, also gave a major metabolite production, especially with the strain Lach5. This clearly demonstrates the interest of testing several sugar sources for secondary metabolite production. Copper sulfate was found to be a moderately effective elicitor as the corresponding metabolite level was practically always inferior to that for glucose.

In conclusion, it appears that Psoralea hairy root and callus lines present an original secondary metabolism compared with the whole plants. Future experiments will study the balance between the coumestrol and the furanocoumarin pathways in Psoralea plants and in vitro cultures. At present, it is not clear if callus and hairy root cultures are still able to synthetize the key enzymes which elaborate furanocoumarins, and whether the synthesis is just repressed or if the corresponding genes are missing. New callus lines have been obtained from species other than P. cinerea with the object of increasing the genetical variability of the whole callus line sample. Enzymatic profiles and comparisons between Psoralea plants and in vitro cultures will be undertaken with the object of explaining the lack of furanocoumarin synthesis by callus and hairy root lines.

Selections from the book: Medicinal and Aromatic Plants VIII (1995).