Intraspecific Genetic Diversity of Cistus creticus L. and Evolutionary Relationships to Cistus albidus L. (Cistaceae): Meeting of the Generations?

Cistus (Cistaceae) comprises a number of white- and purple-flowering shrub species widely distributed in the Mediterranean basin. Within genus Cistus, many taxa are subject to various taxonomic uncertainties. Cistus creticus, a prominent member of the purple-flowered clade, is a prime case of the current taxonomic troubles. Floras and databases approve different species names and utilise different or additional/fewer synonyms. Various intraspecific classification systems based on subspecies or varieties are in use. The inconsistent determination of plant material makes it difficult to compare literature regarding the phytochemical diversity and biological activities of plant material and impedes a systematic utilization of the manifold medicinal properties of C. creticus. In the present investigation, we used DNA sequence data from one nuclear region (ITS) and two chloroplast regions (trnL-trnF, rpl32-trnL) to test the intraspecific genetic diversity of C. creticus and its evolutionary relationships to the closely related C. albidus. The combined DNA data confirmed C. creticus as a rather heterogeneous species that integrates two major evolutionary lineages with clearly different genetic characteristics. The ‘Eastern Mediterranean clade’ seems to represent old and ancestral characteristics. This lineage exhibits a close relationship to the geographically distant C. albidus, expressed by very closely related ribotypes and an interspecifically shared chlorotype. The ‘Western Mediterranean clade’ is characterized by a distinctive ITS polymorphism (co-occurring paralogous ribotypes) and more distantly related chlorotypes. The formation of the genetically complex ‘Western Mediterranean clade’ seems to have involved hybridization and recurrent formation or migration movements.

Comparative Metabolite and Gene Expression Analyses in Combination With Gene Characterization Revealed the Patterns of Flavonoid Accumulation During Cistus creticus subsp. creticus Fruit Development

Cistus creticus L. subsp. creticus (rockrose) is a shrub widespread in Greece and the Mediterranean basin and has been used in traditional medicine as herb tea for colds, for healing and digestive hitches, for the treatment of maladies, as perfumes, and for other purposes. Compounds from its flavonoid fraction have recently drawn attention due to antiviral action against influenza virus and HIV. Although several bioactive metabolites belonging to this group have been chemically characterized in the leaves, the genes involved in their biosynthesis in Cistus remain largely unknown. Flavonoid metabolism during C. creticus fruit development was studied by adopting comparative metabolomic and transcriptomic approaches. The present study highlights the fruit of C. creticus subsp. creticus as a rich source of flavonols, flavan-3-ols, and proanthocyanidins, all of which displayed a decreasing trend during fruit development. The majority of proanthocyanidins recorded in Cistus fruit are B-type procyanidins and prodelphinidins, while gallocatechin and catechin are the dominant flavan-3-ols. The expression patterns of biosynthetic genes and transcription factors were analyzed in flowers and throughout three fruit development stages. Flavonoid biosynthetic genes were developmentally regulated, showing a decrease in transcript levels during fruit maturation. A high degree of positive correlations between the content of targeted metabolites and the expression of biosynthetic genes indicated the transcriptional regulation of flavonoid biosynthesis during C. creticus fruit development. This is further supported by the high degree of significant positive correlations between the expression of biosynthetic genes and transcription factors. The results suggest that leucoanthocyanidin reductase predominates the biosynthetic pathway in the control of flavan-3-ol formation, which results in catechin and gallocatechin as two of the major building blocks for Cistus proanthocyanidins. Additionally, there is a decline in ethylene production rates during non-climacteric Cistus fruit maturation, which coincides with the downregulation of the majority of flavonoid- and ethylene-related biosynthetic genes and corresponding transcription factors as well as with the decline in flavonoid content. Finally, functional characterization of a Cistus flavonoid hydroxylase (F3′5′H) was performed for the first time.

Polyphenol Diversity and Antioxidant Activity of European Cistus creticus L. (Cistaceae) Compared to Six Further, Partly Sympatric Cistus Species

This investigation focused on the qualitative and quantitative composition of polyphenolic compounds of Mediterranean northern shore Cistus creticus and six further, partly sympatric Cistus species (C. albidus, C. crispus, C. ladanifer, C. monspeliensis, C. parviflorus, C. salviifolius). Aqueous extracts of 1153 individual plants from 13 countries were analyzed via high performance liquid chromatography (HPLC). The extracts of C. creticus were primarily composed of two ellagitannins (punicalagin and punicalagin gallate) and nine flavonol glycosides (myricetin and quercetin glycosides, with m-3-O-rhamnoside as the dominant main compound). Differences in the proportions of punicalagin derivatives and flavonol glycosides allowed the classification into two chemovariants. Plants containing punicalagin derivatives and flavonol glycosides were especially abundant in the western and central Mediterranean areas and in Cyprus. From Albania eastwards, punicalagin and punicalagin gallate were of much lesser importance and the predominant chemovariant there was a nearly pure flavonol type. With its two chemovariants, C. creticus takes a central position between the flavonol-rich, purple-flowered clade (besides C. creticus, here represented by C. albidus and C. crispus) and the more ellagitannin-rich, white- or whitish-pink-flowered clade (here represented by C. ladanifer, C. monspeliensis, C. parviflorus and C. salviifolius). The median antioxidative capacity of C. creticus plant material was, with 166 mg Trolox equivalents/g dry wt, about half of the antioxidative capacity of C. ladanifer (301 mg te/g dry wt), the species with the highest antioxidative potential.

Combined effect of hot water dipping and Cistus creticus L. leaf extracts on the storage quality of fresh Valencia oranges

This research was designed to test the combined efficacy of hot water dipping (HWD) and leaf extracts of Cistus creticus L. subsp. creticus on the storage quality of fresh Valencia oranges. Experiments were laid down according to a completely randomised design with 10 different treatments. Five replications, with five fruits in each replication, were used in the current study, and research was designed to continue for 150 days. Quality characteristics were observed with 30-days interval. The applications of this study were (1) Control [dipping in water at 25°C for 5 min]; (2) HWD at 50°C for 5 min; (3) HWD + Additives (A) [Arabic gum (0.2%), potassium sorbate (0.05%), citric acid (0.05%) and vitamin C (0.05%)] at 50°C for 5 min; (4) Additives alone at 25°C for 5 min; (5) 1.0% C. creticus leaf + HWD + A; (6) 1.0% C. creticus leaf + A; (7) 0.5% C. creticus leaf + HWD + A; (8) 0.5% C. creticus leaf + A; (9) 0.5% C. creticus leaf + HWD and (10) 0.5% C. creticus leaf. According to the results obtained, both the HWD and C. creticus, alone or in combination, were effective in preserving orange weight during storage, preventing pathogenic decay, maintaining visual quality and fruit firmness, delaying the reduction in titratable acidity, increasing ascorbic acid content and reducing respiration rate of the orange fruits. Results also suggested that the combined effect of HWD with C. creticus leaf extracts provided the highest efficacy.