Oxocativic acid: the diterpene possibly responsible for the allelopathic activity of Cistus ladanifer
Area of Ecology, Faculty of Science, University of Extremadura, 06071 Badajoz, Spain; e-mail: firstname.lastname@example.org
The exudate of Cistus ladanifer is rich in compounds derived from secondary metabolism, including diterpenes. Previous studies have shown C. ladanifer to have a clear seasonal allelopathic activity, and bioassays with oxocativic acid, one of the major diterpenes in the exudate, have shown it to have phytotoxic activity. The present study was aimed at quantifying oxocativic acid in leaves and litter over the course of a year, and at establishing the relationship with C. ladanifer's allelopathic activity.
To quantify one of the major compounds (oxocativic acid) of the exudate of C. ladanifer in leaves and litter and to determine its involvement in the allelopathic activity of the above mentioned species.
Allelopathy, Cistus ladanifer, diterpene, phytotoxicity.
A classical definition of allelopathy is that given by Rice, 1984: "It is the plant-plant chemical interaction, including microorganisms in the term plant, and both stimulatory and inhibitory effects in the term interaction."
Previous studies have demonstrated that the aqueous extract obtained from Cistus ladanifer leaves inhibits the germination and cotyledon birth of several herbs (Chaves, 1994; Chaves and Escudero, 1997). The first studies on the allelopathic behaviour of this species were aimed at identifying the different compounds that might act as allelopathic agents. It was shown that the compounds present in the leaves with the greatest negative effect on germination include butyric acid, methyl propionate, and cinnamic acid. Others, such as hydroxycinnamic, ferulic, hydroxybenzoic, and oxalic acids, do not inhibit germination, but do affect seedling growth negatively (Chaves et al., 2001a). It is noteworthy that these compounds comprise a minor fraction of the exudates, whereas the aglucone flavonoids and the diterpenes comprise major fraction. Of the flavonoids, it has been shown that 3-O-methylkæmpferol, 3,7-di-O-methylkæmpferol, and 4´-O-methylapigenin may also be involved in the allelopathic behaviour of C. ladanifer, although they affect seedling growth only very weakly (Chaves et al., 2001b; Sosa, 2003). Recently, however, the other major fraction, diterpenes, has been shown to have phytotoxic activity, and the major diterpene is oxocativic acid.
Subsequently these compounds have been shown to be present in the soils in which this species is growing. Laboratory trials have found the germination and growth of different herb species to be inhibited in these soils in a seasonally dependent manner, and that the compounds present in this medium vary qualitatively and quantitatively over the course of the year (Chaves et al., 2003; Sosa, 2003).
The different allelopathic compounds may be incorporated into the soil by volatilization, decomposition of the litter, or secretion by different organs of the plant. The allelopathic activity of C. ladanifer soils is greatest in spring, coinciding with litterfall, and indicating that one of the routes by which the compounds are incorporated into the soil may be through the litter. This would imply that the compounds which are most prevalent in the spring might be responsible for, or at least the most implicated in, the allelopathic activity of C. ladanifer.
Based on the hypothesis that the major constituents of the exudate presenting under controlled laboratory conditions will produce the greatest allelopathic activity, the aim of the present work was to quantify in leaves and litter one of these compounds, oxocativic acid, and to determine its involvement in the allelopathic activity of C. ladanifer.
Samples of leaves and litter were collected from different C. ladanifer populations, thereby avoiding bias in the determination of the oxocativic acid content due to the environmental conditions of any population in particular. Twelve C. ladanifer populations distributed from the SW to the NW of the Iberian Peninsula were selected, and sampling was performed in each of the four seasons. Both leaves and litter were collected from different randomly selected individuals.
Oxocativic acid extraction and assay
One gram of leaf and of litter (one sample per population) was extracted with chloroform (1:2). The chloroform was evaporated off, and the residue was re-suspended in 2 ml of methanol. This fraction was then assayed by HPLC using a Spherisorb 5μ (C-18 of 4.6x250mm) reverse phase analytical column, with water/acetonitrile under gradient conditions.
Since this diterpene is not available commercially, the calibration straight line with which to quantify the amount of oxocativic acid in leaves and litter was constructed with known concentrations of this compound that had previously been extracted from C. ladanifer exudate.
The results are expressed as mg/g d.w.
Wheat (Triticum sp.) seeds were germinated in distilled water in total darkness at 22°C. After 4 days, the coleoptiles were cut into 4 mm sections, and placed into test tubes containing a pH 5.6 buffer of sucrose (20g/l), citric acid (1.02g/l), and potassium phosphate dibasic (2.9 g/l). Different buffer solutions were prepared at concentrations of the compound from 10-3 to 10-6M. Five coleoptiles in 5ml of solution at different concentrations were put into each test tube, and these were placed in a rotor that maintained the culture under continual shaking in a chamber in total darkness at 22°C for 24 hours (Macías, 2000). Their lengths were then measured using the computer program Fotomed (Castellano, 2002).
Figure 1 shows that the activity of this compound at a concentration of 10-3M was high, with an 80% inhibition of wheat coleoptile elongation with respect to the control. At 10-4M, the inhibition was reduced to a half. Coleoptile elongation was not inhibited significantly at the two lowest concentrations.
Figure 1. Effect of oxocativic acid on the growth of wheat (Triticum sp.) coleoptiles. The results are expressed with respect to the control.
Quantitative variation of oxocativic acid in C. ladanifer leaves and litter over the course of the year
Table 1 shows the mean amount of oxocativic acid (mg/g d.w.) in leaves and litter in the twelve selected populations over the course of the year. The results show this compound to have been synthesized in young leaves throughout the year, with greater amounts in autumn and winter. In the litter, however, the amounts were significantly greater in spring and summer than in autumn and winter. Hence, the presence of this compound in the leaves is out of phase with its presence in the litter, and the litter that we found on the soil in spring corresponded to the leaves that were on the plant during the winter. It is probably incorporated into the soil by the decomposition of the litter, or by the action of rain washing it off both the leaves and the litter.
Table 1. Oxocativic acid concentration (mean of twelve populations) in young leaves and litter. The results are expressed in milligrams per gram dry weight
Previous studies showed that the soils associated with C. ladanifer inhibit the germination of herbs, especially in spring. The present work has made it clear that compounds such as oxocativic acid present in the exudate, and with allelopathic activity can pass into the soil via the litter, and can exert their effect there. Since, in the litter the greatest amount of oxocativic acid was found in spring when inhibition in the soils is significantly stronger, one can deduce that this diterpene is probably one of the compounds responsible for the allelopathic activity of this shrub. It is necessary, however, to extend this line of research in order to clarify whether the quantities found are sufficient to exert their effect in such a complex medium as the soil, where other physical, chemical, and biological factors could affect oxocativic acid's activity.
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