Flavonoids from Lotus creticus

One flavanone, euchrestaflavanone A and four isoflavonoids (lupinalbin A–C, and a previously unreported 4′-methyl ether of wighteone) were isolated from Lotus creticus. The structures were elucidated by spectroscopy.     

Nectar Amount, Pollinator Visit Duration and Pollination Success in the Mediterranean Shrub Cistus creticus

We have re-examined the nectar status of Cistus creticus, aplant that was previously thought to be an almost exclusivepollen donor. Although attempts to extract nectar with the microcapillarymethod failed, rinsates of the flower interior yielded considerableamounts of sugar, yet with high interplant variation. Enzymaticmicroassays revealed that secreted sugar is adsorbed onto thesurfaces of the numerous filaments surrounding the nectary,on the dense pubescence of the ovary surrounded by the nectaryand on the yellow base of the purplish-pink, free, obovate petals.The conclusion that C. creticus is an efficient nectar donorwas further strengthened by video recording the behaviour ofbees and by the high frequency of nectar thieves. We proposethat the shallow flower shape and the overheating caused bysolar tracking facilitate evaporative concentration of nectar.Alternatively (or in addition), the morphological proximityof the nectary to the filaments and the ovary pubescence facilitatesadsorption and the capillary action of the cell walls worksantagonistically to glass microcapillaries precluding nectarcollection. Addition of artificial nectar in the field had noeffect on bee visit frequency but it increased visit durationsignificantly and decreased abortion rates. In addition, seedyield was increased, especially under insect-limited conditions.We conclude that C. creticus is a nectar donor, and that nectarvariation in the field may modify pollinator behaviour and affectpollination success and the fitness of the plant. Copyright2000 Annals of Botany Company Cistus creticus, insect behaviour, nectar manipulation, pollination success, visit duration     

Isolation and functional analysis of two Cistus creticus cDNAs encoding geranylgeranyl diphosphate synthase

Cistus creticus ssp. creticus is an indigenous shrub of the Mediterranean area. The glandular trichomes covering its leaf surfaces secrete a resin called “ladanum”, which among others contains a number of specific labdane-type diterpenes that exhibit antibacterial and antifungal action as well as in vitro and in vivo cytotoxic and cytostatic activity against human cancer cell lines. In view of the properties and possible future exploitation of these metabolites, it was deemed necessary to study the geranylgeranyl diphosphate synthase enzyme (GGDPS, EC 2.5.1.30), a short chain prenyltransferase responsible for the synthesis of the precursor molecule of all diterpenes. In this work, we present the cloning, functional characterisation and expression profile at the gene and protein levels of two differentially expressed C. creticus full-length cDNAs, CcGGDPS1 and CcGGDPS2. Heterologous yeast cell expression system showed that these cDNAs exhibited GGDPS enzyme activity. Gene and protein expression analyses suggest that this enzyme is developmentally and tissue-regulated showing maximum expression in trichomes and smallest leaves (0.5–1.0 cm). This work is the first attempt to study the terpenoid biosynthesis at the molecular level in C. creticus ssp. creticus.     

Ultrastructure of Anaulus creticus Drebes & Schulz with special reference to its reduced ocelli

The ultrastructure of the vegetative cell of the centric diatom Anaulus creticus Drebes & Schulz is described. Exhibiting the organic wall component (diatotepum) separated from the siliceous parts of the cell wall this species belongs to the diatom group with so-called split-walls [17]. A most interesting feature of the Anaulus cell is the horn region and its internal structure. As there is no silicified sieve-plate at the horn top but just a big hole sealed only by a thin diatotepum with a hexagonal meshwork, this wall structure is termed a "reduced ocellus". The inner part of the horn region is largely occupied by a conspicuous protoplasmic plug of proteinaceous nature. The possible function of this peculiar structure is discussed as well as new ideas about the role of the labiate process are provided.     

New semisynthetic antimicrobial labdane-type diterpenoids derived from the resin "ladano" of Cistus creticus

The antimicrobial activity of fifteen semisynthetic labdane-type diterpenes derived from the two major natural compounds 3 and 4 of the resin "ladano" of Cistus creticus is reported. The chloroethyl carbamidic esters 15 and 20 showed the strongest antimicrobial activity against Gram(+), Gram(-) bacteria and pathogenic fungi.     

Interpopulation Variability in the Essential Oil Composition of Cistus creticus subsp. eriocephalus from Sardinia

Cistus genus is widespread in the Mediterranean regions with several species and is traditionally known as a natural remedy, but few previous phytochemical researches have been reported on Cistus species growing in Sardinia. The aim of this work was to study the interpopulation variability of the volatiles of C. creticus subsp. eriocephalus to find out the natural chemotypes of this taxon for chemotaxonomic purposes. Plant material was collected from seven wild populations in Sardinia. The oils from aerial part were obtained by hydrodistillation and analyzed by GC/MS enabling to identify 185 compounds. The seven samples showed appreciable variations in their chemical composition. MA was found almost exclusively linear hydrocarbons (85%) and MN presented fatty acids (36.97%) as major fraction. CP and MN had high quantity of non‐terpenic carbonylic compounds (63% and 69%, respectively) and in CP was found also sclareol, a well‐known antimicrobial compound. PCA analysis showed the existence of a high interpopulation variability within the essential oils of C. creticus subsp. eriocephalus growing in Sardinia. For example, MN and CP are very close to each other as CG is close to PM. BN is isolated from the others Cistus populations due to lacking essential oil. These data suggest that the basis of variation in the volatile composition of seven C. creticus subsp. eriocephalus populations depends on hybridization and that the sample without essential oil is the only one no‐hybridized.     

Enhanced UV-B radiation,flower attributes and pollinator behaviour in Cistus creticus: a Mediterranean field study

The aim of this investigation was to examine the reasons for the higher pollination success in Cistus creticus under enhanced UV-B radiation (Stephanou & Manetas 1998). Thus, a selected array of floral attributes as well as the frequency and duration of insect visits were studied in the field under ambient or ambient plus supplemental UV-B radiation, simulating a 15% ozone depletion over Patras (38.3° N, 29.1° E). Video-recording revealed two categories of visitors, i.e. true pollinators (bees) and nectar thieves. The frequency of visits to both control and UV-B treated plants was the same and independent of whether the UV-B tubes were on or off during video recording. UV-B radiation had no effect on gross floral morphology (petal surface area, number of pollen grains, stamens and ovules, optical properties of petals and stamens), yet nectary size was almost doubled. In addition, the duration of insect visits was significantly longer on UV-B treated plants, provided that the UV-B tubes were off during monitoring. The differences were abolished during the part of the day that the tubes were on, indicating that the insects were annoyed by supplemental UV-B radiation. These results are consistent with the nectaries producing larger quantities of nectar, which caused the insects to stay longer on flowers of UV-B treated plants and improved pollination success.     

Enhanced UV-B radiation increases the reproductive effort in the Mediterranean shrub Cistus creticus under field conditions

Seedlings of the Mediterranean shrub Cistus creticus L. were grown in the field under ambient or ambient plus supplemental UV-B radiation (simulating a 15% ozone depletion over Patras, 38.3°W, 29.1°E) for 20 months. During this period, measurements of photosynthetic capacity, photochemical efficiency of PS II, chlorophylls and carotenoids were performed once per season. Supplemental UV-B radiation had no significant effect on these parameters nor on the total, above ground biomass accumulation, plant height and leaf specific mass measured at plant harvest. It was observed, however, that UV-B supplementation increased the number of seeds per fruit as well as mean individual seed mass. As a result, seed number and total seed mass per plant were considerably increased. Germination rates of produced seeds were not affected. We may conclude that C. creticus is a UV-B resistant plant whose competitive ability may be improved by enhanced UV-B radiation through an increase in its reproductive effort and a higher contribution to the seed bank.     

Influence of temperature and salinity on the germination of Lotus creticus (L.) from the arid land of Tunisia

Effects of salinity, temperature and their interactions on the rate and final percentage of germination were evaluated for two populations (Msarref, Oued dkouk) of the invasive glycophyte Lotus creticus Linné, grown under arid environmental conditions of the Tunisia. Seeds that were not treated with NaCl germinated well in a wide range of temperatures. For both populations, maximum germination occurred in distilled water at 25°C and lowest germination for all salinities was at 35°C. Germination was substantially delayed and significantly reduced with an increase in NaCl to levels above 300 mm . Compared to the Oued dkouk population, final germination and germination rate of the Msarref population was completely inhibited at 300 mm NaCl. The interactive effect of temperature and NaCl concentration on final germination and germination rate was significant (P < 0.01), indicating that the germination response to salinity depended on temperature. The inhibition of Oued dkouk population seed germination at high salt concentration was mostly due to osmotic effects while ionic effects were noted at Msarref population. The germination behaviour of the Oued dkouk population would therefore imply adaptive mechanisms to saline environments, while in the Msarref population such mechanisms seem to be absent. Since seed germination is more sensitive to salinity stress than the growth of established plants, the greater tolerance to salinity of Oued dkouk population would be an adaptive feature of this population to saline environment.     

A chemometric interpopulation study of the essential oils of Cistus creticus L. growing in Crete (Greece)

The chemical composition of the essential oils of twenty-five populations of Cistus creticus subsp. creticus L. from the island of Crete (Greece) and their interpopulation variability were analysed in detail by GC-MS. 142 compounds were identified representing an average of 56.8-89.8% of the oil composition. The components are represented here by homologous series of monoterpenes, oxygenated monoterpenes, sesquiterpenes, oxygenated sesquiterpenes, diterpenes, labdane diterpenes, aldehydes, alkanes, esters, fatty acids, ketones, and others. Labdane diterpenes were detected and identified in the essential oils and have been found in high percentage composition. The results from the chemical analysis of the essential oils were submitted to chemometric cluster analysis in order to detect some pattern distribution and to identify which constituents can differentiate the groups of individuals. Two main chemotypes (clusters) were well differentiated; the first deals with eight populations of West Crete and the second with the rest of the populations. Cluster analysis based on labdane type diterpenes patterns, proved to be the best chemotype for the examined populations among the other chemical groups.     

Phytochemical analysis of the labdanum-poor Cistus creticus subsp. eriocephalus (Viv.) Greuter et Burdet growing in central Italy

Phenolic constituents and essential oil from the aerial parts of Cistus creticus subsp. eriocephalus (Viv.) Greuter et Burdet growing in central Italy were analysed by HPLC-MSⁿ and GC–MS, respectively. Furthermore, six constituents were isolated by semipreparative HPLC from the methanol extract and their structures were determined on the basis of 1D and 2D NMR measurements as well as MS spectra. Isolated compounds were one new natural product, i.e. the shikimic acid ester 3,5-diihydroxy-4-(O-β-d-glucopyranosyl)-cyclohex-1-en-1-(O-β-d-glucopyranosyl)-ester (27), and six flavonoid glycosides, namely quercetin-3-O-β-D glucopyranoside (16), quercetin-3-O-rhamnoside (17), tricetin-4′-O-β-D glucopyranoside (24), tricetin-4′-O-β-D rutinoside (21), 3′-methoxy-quercetin-3-O-(3-β-Dglucopyranosyl-2-rhamnopyranosil-4-glucopyranosyl-2-rhamnopyranosil)-glucoside (25) and 3′,4′dimethoxyquercetin-3-O-rhamnopyranoside (26). GC–MS analysis of the essential oil highlighted the occurrence of aliphatic compounds, mainly fatty acids, whereas labdane-type compounds were very scant. Our results showed that C. creticus subsp. eriocephalus has a different chemical profile with respect to the other subspecies due to the lack of labdane derivatives. On the other hand, this subspecies contains several phenolic constituents like ellagitannins, gallotannins and flavonoids, some of which can be of chemotaxonomic value.     

Molecular evidence for hybrid origins of the invasive polyploids Carthamus creticus and C. turkestanicus (Cardueae, Asteraceae)

The hexaploids Carthamus creticus and C. turkestanicus are noxious weeds with wide but non-overlapping Mediterranean distributions, and C. creticus, together with another polyploid, C. lanatus, have also invaded similar climatic regions in North and South America, South Africa and Australia. Here we infer their origins using sequences of the plastid intergenic spacer trnH-psbA and the intron trnK and three introns of nuclear low-copy genes of the RNA Polymerase family (RPD2 and the duplicated RPC2), as well as RAPD markers (random amplified polymorphic DNA). Phylogenetic analyses of the nuclear introns and additivity analysis of the RAPD markers support the hypotheses that the two hexaploids are allopolyploids sharing a tetraploid progenitor lineage represented by the broadly Mediterranean C. lanatus, combined with different diploid progenitor lineages consistent with the different geographic distributions of the hexaploids. Whereas C. leucocaulos from the south-eastern Greek Islands represents the diploid progenitor lineage of the western C. creticus, the Irano-Turanian C. glaucus represents the diploid progenitor lineage of the eastern C. turkestanicus. The plastid data suggest that the diploid lineages served as the maternal progenitors of the hexaploids.     

Cistus creticus subsp. eriocephalus as a Model for Studying Plant Physiological and Metabolic Responses to Environmental Stress Factors

Variations in physiology and metabolic products of Cistus creticus subsp. eriocephalus along an altitudinal gradient (350–750 m.a.s.l.) within the Monti Lucretili Regional Natural Park (central Italy) were studied. The results showed that the phenol production was in relationship with the net photosynthetic rates and the chlorophyll content. In particular, the increasing caffeic acid (CA) content with altitude suggested its role in providing an additional photo‐protection mechanism, by its ability to consume photochemical reducing power and acting as an alternative C‐atom sink under high light conditions. The metabolic production was tested by high performance thin layer chromatography (HPTLC) fingerprint analysis, highlighting the potential of this technique in biologic studies.     

Ecophysiology of fire-stimulated seed germination in Cistus incanus ssp. creticus (L.) Hey wood and C. salvifolius L.

Abstract. Seed germination characteristics were investigated in the most common Cistus species in Greece, namely C. incanus ssp. creticus and C. salvifolius. In addition to the soft seed subpopulation, both species produce a large fraction of hardcoated, water-impermeable seeds which can be softened and, thus, promoted to germinate by mechanical scarification and thermal pretreatment. Temperature and light control of seed germination are unimportant. In the ecological context of the Mediterranean ecosystems, the eventually advantageous, opportunistic strategy of germination is based on: (a) seed heterospermy (which allows the smaller, softcoated fraction to germinate promptly each year while the majority of the seeds, the hard ones, accumulate in the soil); (b) the seed population heterogeneity in relation to coat hardness (so that any heat conditions produced by fire induce the softening and germination of a certain seed fraction); (c) the notably low germination rate (which suppresses premature germination); (d) the wide, Mediterranean-type (relatively cool), temperature range of germination (while higher temperatures simply inhibit but do not induce any dormancy); and (c) the apparent lack of photo-sensitivity (enabling germination under every light regime). In non-fire years, the temporal distribution of field germination and seedling appearance might be partly determined by the seed dispersal strategy of the individual Cistus species. Nevertheless, the post-fire regeneration response is manifested in the form of a huge wave of germination (of practically all seeds softened by the fire heat), shortly after the onset of the rainy season.     

The effects of seasons, exposure, enhanced UV-B radiation, and water stress on leaf epicuticular and internal UV-B absorbing capacity of Cistus creticus: a Mediterranean field study

The seasonal variation in leaf epicuticular and internal (cellular)UV-B absorbing compounds was followed in field-grown shrubsof Cistus creticus L. Fully exposed and permanently shaded (undera canopy of evergreen sclerophylls) individuals were used. Nearmonthly measurements were performed for three years. In addition,a 2-year field experiment was established, in which fully exposedplants received ambient or ambient plus supplemental UV-B radiation,simulating a 15% ozone depletion over Patras (38.3 N, 29.1E). A second, six-month field experiment investigated the effectsof additional summer irrigation on UV-B absorbing compounds.It was found that: (a) exposed plants had considerably higherconcentrations of UV-B absorbing compounds (both epicuticularand internal), irrespective of season, (b) exposed and shadedplants exhibited dramatic and yearly consistent seasonal fluctuationsin epicuticular material with a c. 18-fold difference betweenthe summer maxima and winter minima. Internal UV-B absorbingcompounds showed the same seasonal trends, but with far lesspronounced differences, (c) In the 2-year field experiment withenhanced UV-B radiation, an increase in both epicuticular andinternal UV-B absorbing capacity was evident only during thesummer dry period of the first year. Later on, however, andup to the termination of the experiment, the differences wereabolished, (d) Well-watered and water-stressed plants showedthe same levels of both epicuticular and internal UV-B absorbingcompounds. Although the results indicate that concentrations of epicuticularand, to a lesser extent, internal UVB absorbing compounds fluctuateaccording to the seasonally and site-dependent environmentalstresses, no indications for an effect of water availabilitywere obtained. Therefore it may be concluded that the main determinantof the levels of UVB absorbing compounds is the intensity ofsolar radiation. The relative influence of the various spectralregions of solar radiation cannot be assessed from the presentresults. It was predicted, however, that the effects of increasedsolar UV-B radiation, resulting from the expected ozone depletionover the next decades, will be negligible and probably maskedby the much larger seasonal and site-dependent changes. Key words: Epicuticular, exposure, seasons, UVB absorbing compounds, UVB radiation     

Intra-species variation in transient accumulation of leaf anthocyanins in Cistus creticus during winter: evidence that anthocyanins may compensate for an inherent photosynthetic and photoprotective inferiority of the red-leaf phenotype

Leaf color in some individuals of Cistus creticus turns transiently to red during winter, while neighboring individuals occupying the same site remain green. We have examined whether anthocyanin accumulation can be associated with variations in photosynthetic and/or photoprotective characteristics between the two phenotypes, rendering the red phenotype more vulnerable to photoinhibition and, accordingly, needing additional protection in the form of anthocyanins. Towards this aim, maximum (pre-dawn) and effective (mid-day) PSII photochemical efficiencies, xanthophyll cycle pool sizes and leaf nitrogen contents were seasonably followed, encompassing both the green (spring, summer, autumn) and the red (winter) period of the year. Moreover, the distribution of the two phenotypes in exposed and shaded sites was assessed. The frequency of red individuals was considerably higher in fully exposed sites, pointing to a photoprotective function of leaf anthocyanins. Yet, the assumption was not corroborated by pre-dawn PSII yield measurements, since both phenotypes displayed similar high values throughout the year and a similar drop during winter. However, the red phenotype was characterized by lower light-saturated PSII yields, xanthophyll cycle pool sizes and leaf nitrogen, during both the green and the red period of the year. Based on this correlative evidence, we suggest that winter redness in C. creticus may compensate for an inherent photosynthetic and photoprotective inferiority, possibly through a light screen and/or an antioxidant function of leaf anthocyanins.