Genus Cistus: a model for exploring labdane-type diterpenes' biosynthesis and a natural source of high value products with biological, aromatic, and pharmacological properties

The family Cistaceae (Angiosperm, Malvales) consists of 8 genera and 180 species, with 5 genera native to the Mediterranean area (Cistus, Fumara, Halimium, Helianthemum, and Tuberaria). Traditionally, a number of Cistus species have been used in Mediterranean folk medicine as herbal tea infusions for healing digestive problems and colds, as extracts for the treatment of diseases, and as fragrances. The resin, ladano, secreted by the glandular trichomes of certain Cistus species contains a number of phytochemicals with antioxidant, antibacterial, antifungal, and anticancer properties. Furthermore, total leaf aqueous extracts possess anti-influenza virus activity. All these properties have been attributed to phytochemicals such as terpenoids, including diterpenes, labdane-type diterpenes and clerodanes, phenylpropanoids, including flavonoids and ellagitannins, several groups of alkaloids and other types of secondary metabolites. In the past 20 years, research on Cistus involved chemical, biological and phylogenetic analyses but recent investigations have involved genomic and molecular approaches. Our lab is exploring the biosynthetic machinery that generates terpenoids and phenylpropanoids, with a goal to harness their numerous properties that have applications in the pharmaceutical, chemical and aromatic industries. This review focuses on the systematics, botanical characteristics, geographic distribution, chemical analyses, biological function and biosynthesis of major compounds, as well as genomic analyses and biotechnological approaches of the main Cistus species found in the Mediterranean basin, namely C. albidus, C. creticus, C. crispus, C. parviflorus, C. monspeliensis, C. populifolius, C. salviifolius, C. ladanifer, C. laurifolius, and C. clusii.


INTRODUCTION
Cistus L. (from the Greek word kistos-κίστoς) or rock rose, is a genus of dicotyledonous perennial herbaceous plants that have hard leaves and grow in open areas of stony and infertile soils. They are indigenous to the Mediterranean region and are known for their durability. Even after natural regional forest fires, these plants are capable to grow due to their increased seed germinability after exposure of the seeds to high temperatures (Thanos et al., 1992). In some species seasonal dimorphism is observed, enabling the plants' adaptation to drought conditions, which induces leaves to decrease in size and grow more hair (Aronne and Micco, 2001). The characteristic feature of the genus is a combination of diverse hair types on the leaf, stem, and calyx including non-glandular trichomes. The tufted and stellate as well as the elongate glandular trichomes produce and secrete a resin. In some species (e.g., C. creticus subsp. creticus), this resin is rich in biologically-active and pharmacologicallyinteresting metabolites, such as flavonoid aglycones, glycosides, and terpenoids including labdane-type diterpenes. Since the original description of the genus in 1753 by Linnaeus, a number of species and subspecies have been categorized within Cistus. After several taxonomic re-evaluations, about 21 species of Cistus are now recognized spreading within the white and pink-flowered lineages (Guzmán and Vargas, 2005). Cistus species are distributed both in the eastern and western Mediterranean, where the highest diversity is observed, and are also widespread in the Balearic and Canarian islands Vargas, 2005, 2010). Several of them have been employed in Mediterranean folk medicine as herbal tea infusions for healing digestive problems and colds, as extracts for the treatment of diseases, and as fragnances. The resin, ladano, produced by C. creticus in Crete, Greece and Cyprus, and C. ladanifer in Spain, is exported to a number of Arabic countries where it is used as insence.
In the past 20 years, research on Cistus was of chemical, biological, and phylogenetic nature. Added to this list are the recent genomic and molecular studies. Metabolomic analyses using chromatographic and spectroscopic tools allowed the identification of several chemical groups with distinct biological activities. Among the most important compounds are terpenoids, including diterpenes, labdane-type diterpenes and clerodanes, phenylpropanoids, including flavonoids and ellagitannins, several groups of alkaloids and some other secondary metabolites.
In this review we focus on the major representatives of Cistus species: C. albidus, C. creticus, C. crispus, C. parviflorus, C. monspeliensis, C. populifolius, C. salviifolius, C. ladanifer, C. laurifolius, and C. clusii, which are commonly found in the Mediterranean basin. Due to their plethora of uses and potential valuable therapeutical activities they have been extensively studied.

SYSTEMATICS OF CISTUS SPECIES
The family Cistaceae (Angiosperm, Malvales) consists of 8 genera (Arrington and Kubitzki, 2003) and 180 species, with 5 genera native to the Mediterranean area (Cistus, Fumara, Halimium, Helianthemum, and Tuberaria). The taxonomic separation of the genus is based on phenotypic observations, including morphological characters like shape, nerve number, color and trichomes of leaves and stems, and reproductive characteristics such as petal and sepal number, shape and color of flowers, number of fruit valves and style size. The phenotype-based genus taxonomy was confirmed recently using plant chemotype and molecular approaches.
More recently, Demoly and Montserrat (1993) described the distribution of 12 species of genus Cistus that grow in Iberia. In this approach, 3 subgenera were classified: I. subgenus Cistus, containing C. albidus, C. creticus, C. crispus, and C. heterophyllus; II. subgenus Leucocistus, containing Ledonia with species C. monspeliensis, C. salviifolius, C. psilosepalus, and C. populifolius, and section Ladanium with C. ladanifer and C. laurifolius; and III. subgenus Halimioides containing C. clusii and C. libanotis. From this study, it became apparent that most Cistus species grow in western Mediterranean. The same conclusion was reached for the species distribution (Table S1), where the main 10 Cistus species, discussed in this review, are distributed in 28 areas. Specifically, numerous species grow in Spain followed by Morocco, Italy, Portugal, Algeria, and France. Conversely, in the eastern Mediterranean the number of species is low with the most widespread species being C. creticus, C. palviflorus, and C. salvifolius.
A recent classification of Cistaceae is based on combined nuclear (ncpGS, ITS) and plastidic (trnL-trnF, trnK-matK, trnS-trnG, rbcL) DNA sequence comparisons, which divided Cistus into 3 subgenera (similar to Demoly and Montserrat, 1993): the purple flowered subgenus Cistus and the white flowered subgenera Leucocistus and Halimioides (Figure 1) (Guzmán and Vargas, 2005;. Interestingly, C. palviflorus appeared most closely related to subgenus Leucocistus (white flowers), although it possesses light purple flowers. Similar observations led in the past to the creation of a separate section for C. palviflorus, namely Ledonella. In another work, the evolution of family Cistaceae was studied by the phylogenetic analysis of plastid rbcL and trnL-trnF sequences ). This study confirmed the clear separation of the genus into 2 groups, with purple (excluding C. palviflorus) and white flowers FIGURE 1 | Classification of three subgenus of Cistus genus based on analysis of trnF, matK and ITS sequences (Guzmán and Vargas, 2005) and plastid rbcL and trnL-trnF sequences ). and certified the family Cistaceae as monophyletic, sisterly to families Dipterocarpaceae and Sarcolaenaceae . A similar classification was achieved by analyzing polyphenolic composition of aerial parts of the most common species, which separated Cistus subgenus from the two other subgenera by its higher flavonoid content (Barrajón-Catalán et al., 2011).
Another phylogenetic study confirmed the chemical and genetic (ISSR-PCR amplification) differentiation between the C. creticus subspecies eriocephalus and corsicus . The affinity and distance estimation between individual plants of C. creticus L. in Corsica and Sardinia, as inferred by the trn-F and RPL32-TRNL sequences of cpDNA, showed that the plants were divided into 4 groups having evident correlation with the region . In another taxonomic approach the composition of essential oils of C. salviifolius in 15 Cretan (Greece) populations was studied, dividing the plants into 3 groups, most of them belonging to the group with high camphor production (Demetzos et al., 2002b). Also, a chemometric interpopulation study of C. creticus in the East and West parts of Crete showed the existence of a high variability within the essential oils. The plants were geographically divided into three groups, two in the West and one in the Eastern part of Crete (Demetzos et al., 2002a).

BOTANICAL CHARACTERISTICS GENUS CISTUS L
Cistus plants are small, woody shrubs with a straight stem that has opposite rich-spreading branches and can reach an average of one meter in height (Sweet, 1830). On branches grow usually corrugated leaves, simple and indivisible, either petiolate or sessile, growing opposite or alternate, and carry simple epidermal hair (trichomes) either asteroid or in bunches. Two types of trichomes appear in Cistus, the non-glandular or stellate and the glandular which secrete a resinous exudate, the ladano, to which they owe their distinctive aromatic scent (Gulz et al., 1996). The plants have a terminal or axillary cymose inflorescence, in some species racemose or umbel, with unilateral scorpioid cyme, and by reduction solitary flowers (Demoly and Montserrat, 1993). The flowers are ephemeral, stimulated by the morning light, have either white or pink/purple petals and 3-5 petals. There are numerous stamens, while the ovary has 5 carpels (but can also be 6-12), the style is straight, usually long and inconspicuous, and the stigma is large, discoid with 5-12 lobes (Demoly and Montserrat, 1993). Cistus plants have numerous polyhedral seeds with two linear cotyledons. Their chromosome number is n = 9 (2n = 18) (Demoly and Montserrat, 1993). The plants form ectomychorrizal roots through strict symbiotic associations with various mychorrizal fungal species, mainly belonging to genus Lactarius (Comandini et al., 2006). These are characterized by a multilayered mantle and a Hartig net hyphae physiology that involves both epidermal and cortical cell layers (Comandini and Rinaldi, 2008).
The specific botanical characteristics such as are color of petals, number of sepals, and fruit compartments, type of leaf base and size of the styles are the most commonly used in Cistus systematic classification (Table S2).
Their general morphological characters as well as their adaptability mechanisms to various harsh environmental conditions will be briefly discussed.

Subgenus I: Cistus L
Flower morphology is characteristic in this subgenus. Specifically, each flower consists of five sepals, with pink or purple petals, 80-150 stamens with exine, rugulate pollen about 1.4 μm thick, a long style similar or exceeding the stamens in height with polyspermous placentas (Demoly and Montserrat, 1993).
Within section Erythrocistus, resin excreting glandular trichomes appear in C. albidus, C. creticus subsp. creticus, the short, curled-leaved C. crispus (Gulz et al., 1996) and C. parviflorus. Peculiarly, C. creticus subsp . eriocephalus leaves seem to contain only non-glandular trichomes ). This can explain the different chemical profile between the two subspecies, C. creticus subsp . creticus (CC) and C. creticus subsp . eriocephalus (CE). This observation can be of valuable help in applying modern genomic approaches that allow the distinction between the two sub-species in order to isolate and characterize biosynthetic genes leading to the production of active labdane diterpenes present in the former sub-species.
C. albidus has bright purple flowers (June to August), C. creticus purplish-pink (mid-April to mid-June) and so are those of C. crispus (June to August), while C. parviflorus has small light pink flowers (Sweet, 1830).
C. albidus displays ecotypic differentiation, at least when growing in semi-arid climates, being able to adapt the growth of its branches and leaf dimensions, acquiring the greatest growth under plentiful water availability, while slowing growth and tending to phenotypically converge under drier environments (Grant et al., 2005).
The leaves of the Cretan rock rose C. creticus exhibit the phenomenon of seasonal dimorphism, as an adjustment mechanism for acclimatization to the Mediterranean climate. During summertime, when water is limited, brachyblasts are developed that have leaves five-times shorter than the ones in winter, with stomata located abaxially inside crypts (Aronne and Micco, 2001). During wintertime, the newly developed dolichoblasts are fourteen times longer, bearing a bigger number of leaves with stomata distributed across the lower surface.
The curled-leaved C. crispus reaches only up to 70 cm in height (Sweet, 1830) and is covered by whitish hair (Sweet, 1830). C. parviflorus on the other hand, is one of the tall Cistus plants, that can reach a height of 1.5 m. According to , C. parviflorus is actually classified within the white flowered lineage of the C. salviifolius species group.
In section Ledonia belong the two most widespread Cistus species, C. monspeliensis and C. salviifolius, together with C. populifolius. Characteristics of this section are the five sepals, which are either subequal or the two external are longer, and the style being slightly shorter than the stamens (Demoly and Montserrat, 1993). Both C. monspeliensis and C. salviifolius carry glandular and non-glandular trichomes, while distinctive to C. populifolius is the existence of only the glandular type (Gulz et al., 1996). C. populifolius is further divided in subspecies populifolius and major Dunal.
C. monspeliensis, also known as the Montpelier rock-rose, is characterized by its aromatic leaves and its small white flowers (Angelopoulou et al., 2001a;Kalpoutzakis et al., 2003). It also exhibits seasonal leaf dimorphism with alternating wide and thin late autumn/early winter and thicker late spring/early summer leaves with larger trichome density, while both types coexist on the same plant during early spring (de Dato et al., 2013). Summer leaves have high leaf mass area and tissue density, low leaf surface area and thick adaxial cuticle, traits that contribute to the plants endurance to drought conditions and resistance to fire (Catoni et al., 2012). As expected, long-term experimental drought conditions during the transition to summer leaves can have significant effect on leaf functioning (de Dato et al., 2013). In a relevant study, over-imposed drought resulted in early leaf litter and a reduction of spring-leaf lifespan, thus a shorter vegetative season, which can have a negative effect on C. monspeliensis' survival in Mediterranean shrubland (De Dato et al., 2008;de Dato et al., 2013).
Characteristics of section Ladanium include three sepals, large petals and an inconspicuous style (Demoly and Montserrat, 1993). It consists of the laurel-leaved C. laurifolius, which is the tallest (1-2 even 3 m) and the very short (50-400 cm) flat-leaved Gum C. ladanifer, which is further divided in three subspecies, ladanifer, africanus Dans, and sulcatus Demoly. Both species carry non-glandular and resin producing glandular trichomes on the abaxial surface of the leaves (Gulz et al., 1996). In C. ladanifer, wavy lamina-forming crypts are arranged on the abaxial side, near which non-glandular trichomes are mostly gathered (Tattini et al., 2007).
C. clusii, also called Clusius's rock rose, is a vigorously growing shrub, highly resistant to drought (Pugnaire and Lozano, 1997). It belongs to the C. clusii group, comprising one of the two white flowered Cistus lineage species, together with C. munbyi, and is divided in two subspecies, namely subsp. clusii and subsp. multiflorus Willk ).

GEOGRAPHICAL DISTRIBUTION GENUS CISTUS L
Cistus plants are extensively distributed in the Mediterranean region, covering most areas from the Canary Islands and Madeira to Caucasus and Israel, colonizing the Iberian, Apennine, Balcan, Crimean, and Anatolian peninsulas and North Africa. In this review we have studied the ten most prominent Cistus species, which are widely spread within the Mediterranean basin (Table  S1). The geographical distribution of the species and subspecies can be further explored by visiting the following webpage: Interactive Map of Cistus distribution. In the next paragraphs we describe the distribution, diversification and habitat preference of these species.

Subgenus I: Cistus L
C. albidus grows in evergreen shrublands, is partially droughtdeciduous (Grant et al., 2005) and a non-strict calcicole (Soriano and Gómez Miguel, 2009) that prefers calcareous and basic soils (De Vega et al., 2008) and woodlands with plenty Pinus and Quercus compost, in dry areas with altitude up to 1200 m (Guzmán and Vargas, 2010). Phylogenetic studies have shown that it is closely related to the endemic species forming the Canarian Cistus lineage, while still forming a monophyletic group with two other purple-flowered species exclusive in the Mediterranean basin, C. creticus and C. heterophyllus (Guzmán and Vargas, 2010).
Several populations of C. creticus are spread in central-eastern Mediterranean, including Corsica and Sardinia  and the island of Crete in Greece (Demetzos et al., 2002a). Among the three C. creticus subspecies identified, subsp. corsicus is limited to the islands of Corsica and Sardinia ). More than twenty five populations of subsp. creticus are endemic to the coastal areas of Crete (Greece) (Demetzos et al., 2002a). Subspecies eriocephalus is exclusive to the Mediterranean area , mainly located on the islands of Corsica, Sardinia , and Crete (Demetzos et al., 1997).
C. crispus is endemic to southern France, Spain, Iberian, and Apennine Peninsulas and to northwest Africa ) and grows in clay or stony soils.
The purple-flowered C. parviflorus is a distinctive member of the white-flowered species lineage of Cistus that seems to have diverged in the Middle Pliocene (3.13 ± 0.08 Ma) (Guzmán and Vargas, 2010). Though it appears that early divergence of Cistus species occurred in the western Mediterranean, C. parviflorus is distributed exclusively in the eastern Mediterranean Guzmán and Vargas, 2010). It prefers dry climates and shrublands with calcicolous soils ).

Subgenus II: Leucocistus WILLK
Species of Leucocistus are widespread in the Mediterranean basin and Madeira, the Canary and Balearic Islands, reflecting their successful adaptation and colonization in Mediterranean habitats (Robles and Garzino, 2000;Guzmán and Vargas, 2005).
C. monspeliensis is spread from the western Mediterranean to the Canary Islands and Madeira where it seems to have occurred naturally without any human intervention (Guzmán and Vargas, 2010). The origin of this colonization and further diversification in the Canaries appears to be on the islands of Tenerife and Gran Canaria that have favorable ecological conditions for the growth of the species, and where C. monspeliensisis is widely distributed today (Fernández-Mazuecos and Vargas, 2011). Comparative phylogeography was used to demonstrate that the migration of the species to La Gomera and El Hierro occurred via long-distance dispersal from Tenerife to the southwest. C. monspeliensis is dominant in evergreen garrigue vegetation, inhabiting acidic, limestone, silicolous and calcareous hills and colonizing areas that are rich in Quercus and Pinus trees compost or have been disturbed by fire (Angelopoulou et al., 2001a;Guzmán and Vargas, 2005;Catoni et al., 2012). It has also been demonstrated to be a non-strict calcifuge (Soriano and Gómez Miguel, 2009).
C. populifolius inhabits areas of the western Mediterranean basin and prefers volcanic and silicolous soils .
C. salviifolius is the most widely spread species of the genus Cistus around the Mediterranean basin. At least three intercontinental colonizations are responsible for its wide distribution, leading to little geographical isolation with high genetic diversity within populations, but no genetic differentiation between the different populations of C. salviifolius (Farley and McNeilly, 2000;Fernández-Mazuecos and Vargas, 2010). The factors that caused the dispersion of C. salviifolius around the Mediterranean were mostly ecological, such as the climate and the soil. It grows in silicolous and calcicolous soils and occurs on sandy soils of a wide range of habitats , while it is often located within the understorey in wooded areas (Farley and McNeilly, 2000).

Section 2: Ladanium (SPACH).
Natural habitats of C. ladanifer are located exclusively in the western Mediterranean. The subspecies of C. ladanifer are distributed in close and overlapping geographical regions. It grows in volcanic and silicolous soils in habitats with dry and hot climate .
The adaptation of this species in dry, hot areas is due to its hairy, amphistomatous, and wavy leaves with stomata mostly concentrated in the crypts formed on the abaxial surface of the leaf (Tattini et al., 2007).
C. laurifolius prefers silicolous soils and mesic and high altitudes with Mediterranean mountain climate. This ecological preference for habitat has isolated the European and African populations, which were produced by a single, eastward migration event (Fernández-Mazuecos and Vargas, 2010).

Subgenus III: Hamilioides WILLK
The species in this subgenus are exclusive in the western Mediterranean (Guzmán and Vargas, 2005).
C. clusii is highly efficient in surviving in harsh environments colonizing post-fire and perturbed areas (Pugnaire and Lozano, 1997). It prefers calcicolous soils and dry to semi-arid environments, and can grow in high altitudes, up to 1500 m from the coastline ).

TEPRENES
Metabolite content and volatiles in Cistus are influenced by several factors including diurnal, seasonal, ecological, drought, temperature, plant age, and precipitation. Also, depending on the type of trichomes they contain, Cistus species can be high producers of monoteprenes and sesquiterpenes, while others in diterpenes and clerodanes.
The monoterpene phenol carvacrol was a major constituent of Tunisian C. monspeliensis leaves and essential oils, while other polyphenolic compounds such as diisobutyl ester (phthalic acid) and benzyl benzoate were also strongly represented Loizzo et al., 2013). Moreover, carvacrol and αterpineol were the only monoterpene compounds isolated from leaf essential oil of C. monspeliensis plants found in Crete (Greece) (Angelopoulou et al., 2001a(Angelopoulou et al., , 2002, while several phenolic acid derivatives were identified in plants grown in Spain ( Barrajón-Catalán et al., 2011). Several monoterpenes, including hydrocarbons and oxygenated monoterpenes were identified as the major components in leaves and essential oils of Tunisian and French C. monspeliensis plants (Rivoal et al., 2010;Jemia et al., 2013;Loizzo et al., 2013). Monoterpenes were 14.47% of the total essential oil extracted from Cretan C. parviflorus (Demetzos et al., 1990b). The highest concentration of monoterpenes in the essential oil from C. parviflorus plants grown in Crete were mainly oxygenated monoterpenes while no traces of monoterpene hydrocarbons were reported. Carvacrol was identified as the major constituent in all samples (Angelopoulou et al., 2001b). Mostly oxygenated monoterpenes and only a small fraction of monoterpene hydrocarbons have been detected in a large number of C. salviifolius populations from Crete (Greece) and Tunisia (Demetzos et al., 2002b;Loizzo et al., 2013).
High concentrations of sesquiterpenes have been measured in aerial parts and essential oils of several C. ladanifer populations in France, Portugal, Spain and Morocco, with vidiflorol being the most abundant molecule (Mariotti et al., 1997;Ramalho et al., 1999;Robles and Bousquet-Mélou, 2003;Gomes et al., 2005;Teixeira et al., 2007;Zidane et al., 2013). Analysis of essential oil composition of C. monspeliensis grown in Tunisia led to the identification of accountable amounts of oxygenated sesquiterpenes and sesquiterpene hydrocarbons (Loizzo et al., 2013). Another study conducted with plants at the same site detected a large variety of sesquiterpenes but at low concentrations . Several sesquiterpenes were also isolated from C. monspeliensis plants naturally growing in areas of France and Greece (Robles and Garzino, 2000;Angelopoulou et al., 2001aAngelopoulou et al., , 2002Rivoal et al., 2010). Sesquiterpene compounds identified in C. monspeliensis plants located in Crete (Greece), accounted for a total content of 38.5% in leaf and 6.6% in fruit essential oil (Angelopoulou et al., 2001a).
Mainly oxygenated sesquiterpenes were identified in high percentages in the essential oil of nine populations of C. parviflorus endemic to the island of Crete, with caryophyllene oxide and α-epi-cadinol being the most abundant (Angelopoulou et al., 2001b). High concentrations of sesquiterpenes were also found in aerial parts and essential oils of several C. salviifolius Cretan and Tunisian populations, among which oxygenated sesquiterpenes were the most abundant (Demetzos et al., 2002b;Loizzo et al., 2013).

PHENYLPROPANOIDS
Flower scent is a vital strategy that plants use for attracting pollinators and ensuring their reproduction and survival (Gang, 2005). Volatile phenylpropanoids have a significant role among the compounds emitted by the plants in order to contribute to this aroma. Moreover, as a defensive mechanism of the plants against high solar radiation and drought, the content of the antioxidant flavonoids in Cistus species is highly variable. In most species, increased concentrations of flavonoids are observed during summer and in younger leaves. In addition to emissions, light, especially UV-B radiation, positively influences in a periodic manner the absorbing capacity of epicuticular phenolic substances in C. creticus, contributing to the plants photoprotective mechanism (Stephanou and Manetas, 1997). High concentrations of the non-volatile group of tannins have also been measured in various Cistus species, including the hydrolysable gallic and ellagic acids and ellagitannins (Barrajón-Catalán et al., 2011).

FATTY ACIDS
Several fatty acids were identified in aerial parts and essential oils of C. albidus growing in north-eastern Spain, including tetradecanoic acid and pentadecanoic acid (Llusià et al., 2010;Müller et al., 2014). Fatty acid composition of C. albidus seeds was studied in both young and old plants. High concentrations of linoleic acid, and generally polyunsaturated as well as very long chain saturated fatty acids were found in seeds of the older plants (Müller et al., 2014). Various fatty acids and esters were identified as minor or major components of aerial parts and essential oil content of French, Greek, and Tunisian C. monspeliensis, C. creticus subsp. creticus, and C. salviifolius plants (Demetzos et al., 1994b(Demetzos et al., , 2002bRobles and Garzino, 2000;Angelopoulou et al., 2001a;Jemia et al., 2013;Loizzo et al., 2013).

PHYTOHORMONES AND VITAMINS
The production of phytohormones and vitamins were studied in C. albidus seeds (Müller et al., 2014), leaves, and flowers (Oñate and Munné-Bosch, 2010), in relation to plant maturity. The main vitamin E compound in C. albidus seeds was α-tocopherol, whose content was higher in mature plants than the younger ones (Müller et al., 2014). Similarly, seeds of mature plants had higher concentrations of both salicylic acid and jasmonic acid, which was not the case in flowers and leaves, where no concentration differences were observed between the age groups (Oñate and Munné-Bosch, 2010). The adaptability of the species to drought involved an increase in abscisic (ABA) and ascorbic acid (AA) levels, as well as leaf H 2 O 2 concentrations, localized mainly in mesophyll cell walls, xylem vessels, and differentiating sclerenchyma cells (Jubany-Marí et al., 2009). Recovery from drought implicated the readjustment of ABA, AA, and H 2 O 2 to their basal concentrations (Jubany-Marí et al., 2009). Moreover, efficient acclimation to drought was only achievable in the first of three consecutive cycles of experimental drought and re-watering applications of C. albidus plants (Galle et al., 2011).
C. creticus is also a highly drought-resistant plant, and was used as a model to study drought-induced changes in the expression of genes encoding enzymes involved in isoprenoid biosynthesis, as well as the corresponding metabolites (chlorophylls, carotenoids, tocopherols, and abscisic acid) and endogenous concentrations of other growth regulators (jasmonic and salicylic acids, JA and SA, respectively) (Munné-Bosch et al., 2009). Among the genes studied, those encoding homogentisate phytyl-transferase (HPT) and 9-cis-epoxycarotenoid dioxygenase (NCED) were induced even at early stages of drought, and were strongly correlated to the levels of the corresponding metabolites. The simultaneous increase in concentrations of ABA and α-tocopherol (but not JA and SA), led the authors to suggest that the genes encoding HPT and NCED may play a key role in drought stress resistance by modulating ABA and tocopherol biosynthesis ).

ANTIBACTERIAL, ANTIFUNGAL
Organic and aqueous leaf extracts of C. monspeliensis, and also C. villosus (=incanus), growing naturally in Morocco and Tunisia were shown to have antimicrobial and antifungal properties that were mostly active against Staphylococcus aureus, Enterococcus hirae, and Pseudomonas aeruginosa and the yeast Candita glabrata (Bouamama et al., 2006). Flower extracts of C. monspeliensis were active against gram-positive bacteria species of genus Staphylococcus and had significant growth-inhibitory effects on Staphylococcus epidermis (Sassi et al., 2007). Furthermore, a cisclerodane diterpene isolated in large amounts and characterized from C. monspeliensis was very active against Staphylococci species and had four times higher activity than the labdane-type diterpene sclareol (Kolocouris et al., 2001).
Antimicrobial properties have been demonstrated for several phenolic monoterpenes, such as thymol and carvacrol, and other carbonylic and phenolic compounds identified among C. creticus and C. albidus volatiles (Maccioni et al., 2007;Hutschenreuther et al., 2010). Phenolic compounds present in extracts from C. ladanifer aerial tissues displayed antifungal activity against Candida species (Barros et al., 2013) in a dose-dependent manner . Strong antimicrobial activity against gram-positive bacteria was also demonstrated for C. ladanifer, and against gram-negative bacteria for C. populifolius .

ANTIVIRAL
The polyphenol rich extract CYSTUS052 derived from C. incanus was shown to exhibit potent anti-influenza virus activity without causing toxic side effects or inducing viral resistance . The first clinical study showed that C. incanus extract (CYSTUS052) could be applied for the treatment of upper and lower respiratory tract infections (Kalus et al., 2009). Moreover, C. ladanifer and C. populifolius extracts were able to inhibit the replication of the vesicular stomatitis virus (VSV) (Abad et al., 1997).
Plant-derived remedies for human use need to be carefully prepared in order to result in active antioxidant substances. Indeed, C. incanus beverages exhibit decreased amounts of phenolic substances and reduced antioxidant activity if an incorrect selection of brewing process parameters (brewing water, temperature, and duration) is made (Riehle et al., 2013).

CYTOTOXIC/ANTICANCER
Cytotoxic activity of shoot extracts from an in vitro culture of C. creticus subsp. creticus against human HeLa cells was shown (Skorić et al., 2012). Shoot extracts (rich in labdane diterpenes) were able to exert cytotoxic activity on HeLa (cervix), MDA-MD-453 (breast), and FemX (melanoma) cancer cells. Cytotoxic and antitumor activities of the bioactive, highly lipophilic, and naturally produced labdane-type diterpenes was improved by incorporating them in liposomal formulations, a process that constitutes these compounds suitable for testing in in vivo experiments (Kyrikou et al., 2005;Matsingou et al., 2005;Hatziantoniou et al., 2006). The labdane-type diterpene sclareol, which is used today as a certified drug, has antitumor activity against human breast cancer cell lines and enhances the activity of known anticancer drugs . Sclareol is activated through a p53-independent mechanism that targets the G 1 phase of the cell cycle and therefore apoptosis of human cancer cells is induced through the activation of caspases (Mahaira et al., 2011).
Three flavonoids were also tested against eleven leukemic cell lines. Myricetin had no activity, a myricetin ether that was isolated from the hexane extract of C. monspeliensis exhibited significant cytostatic and cytotoxic activities, and its 3 ,5-diacetyl derivative, which was chemically synthesized, had lower cytotoxic activity (Dimas et al., 2000).

MYORELAXANT
Cistus extracts have been traditionally used in Mediterranean countries for the treatment of diarrhea, peptic ulcers and as antispasmodic agents. Myorelaxant effects of C. incanus and C. monspeliensis aqueous extracts were illustrated on strips of longitudinal smooth muscle of rat ileum and aorta in a concentration-dependent and reversible manner (Attaguile et al., 2004). The authors attributed the inhibition of intestinal mobility by polyphenolic compounds to the high concentrations of flavonoids produced by Cistus plants (Attaguile et al., 2004). Moreover, C. populifolius and C. ladanifer aqueous extracts were evaluated in vitro on animal models and their significant, dosedependent spasmolytic (Sánchez de Rojas et al., 1995) and analgesic effects (de Andrés et al., 1999) were demonstrated. TOXICITY Some secondary metabolites produced by Cistus sp. exert toxic effects on mammals. The most toxic compounds are gallic acid and tannins, which are detrimental to liver and kidneys. Several cases of lethal toxicoses in cattle have been reported, caused by ingestion of Cistus sp., including C. salviifolius (Yeruham et al., 2002). Convulsions and lipofuscinosis in the central nervous system have been reported in sheep as directly linked to grazing on Cistus sp. (Riet-Correa et al., 2009). In addition, flavonoid-rich extract of C. laurifolius was responsible for degenerative effects on the periferic nerval system causing convulsive syndrome in mice (Bregante et al., 1981).
As discussed earlier in this section, many secondary metabolites produced by Cistus sp. exhibit toxicity and therefore can be used in low concentrations in order to exert their biological functions. Direct consumption of the plants or their extracts (as tea infusions) in larger amounts can become harmful and even lethal.

BIOSYNTHESIS OF COMPOUNDS OF INTEREST BIOSYNTHETIC PATHWAY FOR TERPENES INCLUDING LABDANE-TYPE DITERPENES
Isoprenoids are one of the largest classes of metabolites with more than 50,000 representatives identified to date in existing organisms with a central role in both primary and specialized metabolism (Thulasiram et al., 2007). Despite the fact that the building block of all isoprenoids is a C5 isoprene unit, they exhibit an extensive diversity in terms of their size and structure that reflects a wide diversity in their physiological roles. Isoprene is the smallest known 5-carbon terpene, a volatile emitted by the foliage of many tree species. Isoprene emission is considered to enhance tolerance against heat stress and to confer resistance to reactive oxygen species. Monoterpenes are C10-carbon, highly volatile terpenes that contribute to plant odors; they are among the main constituents of flower aromas with an essential role in pollinator attraction. Sesquiterpenes (C15) are also volatile compounds known to play a signaling role in plant defense mechanisms, directly as repellants for herbivores or indirectly through the attraction of herbivore predators and/or pathogens. Diterpenes (C20) are produced by plants for defense purposes (phytoalexins), in addition to serving as precursors of plant hormones (e.g., tocopherols and gibberellins). Triterpenes (C30) are synthesized by the head to tail condensation of C15-carbon terpenes and they are the precursors of phytosterols, brassinosteroids, phytoalexins and waxes. Carotenoids are C40-carbon terpenes that contribute to the pigment of flowers and fruit and play an essential role in pollination and seed dispersal as well as protection against UV light. Higher orders of isoprenoids, the polyprenyls, are used for co-translational modification of proteins called prenylation that promotes their interaction with cell membranes.
There are two distinct pathways responsible for the biosynthesis of terpenes ( Figure S1) in the plant cell (Vranová et al., 2012). The mevalonate pathway (MVA) that operates in the cytosol and starts from acetylCoA, and the 2-C-methyl-d-erythritol 4phosphate (MEP) pathway that operates in the plastid and is initiated by C-5 sugars. Their names come from intermediate precursors of the pathways; the mevalonate in the cytosolic pathway and the 2-C-methyl-d-erythritol 4-phosphate in the plastidial pathway. The final product of both metabolic routes is isopentelyl pyrophosphate (IPP), the universal precursor of all isoprenoids produced in the plant cell. IPP gets isomerized to dimethylallyl pyrophosphate (DMAPP) by the enzyme isopentenyl pyrophosphate isomerase.
In both cytosolic and plastidic compartments, IPP and DMAPP are used by prenyltransferases to produce prenyldiphosphates, the universal precursors of all isoprenoids. A head to tail condensation of IPP and DMAPP results in the formation of geranyldiphosphate (GPP) or neryldiphosphate (NPP), the trans and cis prenyldiphosphate precursor of monoterpenes, respectively. Sequential addition of one more molecule of IPP by farnesyl-diphosphate synthase results in the formation of all-trans or cis-trans farnesyldiphosphate (ee-FPP and zz-FPP), the precursors of sesquiterpenes. For the synthesis of the precursor of diterpenes, geranylgeranyldiphosphatase (GGDP), the addition of one more molecule of IPP is required that is catalyzed by the prenyltransferase geranylgeranyldiphosphate synthase. In C. creticus subsp. creticus two genes that encode active GGDP synthases, CcGGDPS1 and CcGGDPS2, were cloned and functionally characterized (Pateraki and Kanellis, 2008), as also the DXR cDNA from trichomes (Pateraki and Kanellis, 2004).
The prenyldiphosphate precursors are converted to the basic terpene skeletons through the activity of a class of enzymes called terpene synthases. In the lower orders of terpenes these enzymes are further categorized into monoterpene, sesquiterpene and diterpene synthases. The basic skeletons are often further processed by a variety of enzymes including acyltransferases, hydroxylases and dehydrogenases to produce the thousands of different terpenes encountered in nature. There are both cyclic and acyclic terpenes whose exact structure is determined by the specific activity of terpene synthases and the subsequent modifying enzymes. Terpene synthases are classified into two groups based on their catalytic mechanisms: class I and class II reviewed in Chen et al. (2011). In class I enzymes, the prenyldiphosphate precursor is ionized resulting in the formation of a carbocation intermediate, which undergoes cyclizations, hydride shifts and other rearrangments that result in the formation of the basic terpene skeletons of the products. Depending on these rearrangments, some class I terpene synthases specifically give rise to a single product, while others are characterized as multiproduct enzymes. This class of terpene synthases includes primarily monoterpene and sesquiterpene synthases and some diterpene synthases. Class II terpene synthases catalyze a protonation-induced cyclization of the prenyldiphosphate precursor. The reaction is terminated either via deprotanation or nucleophile capture. Characteristic enzyme of this category is the copalyldiphosphate synthase responsible for the synthesis of copalyldiphosphate, the intermediate precursor of ent-kaurene and several labdane-type diterpenes.
Most diterpenes are cyclic and their carbon-rings are formed by two different mechanisms. One is similar to that of class II monoterpene and sesquiterpene synthases. Examples of such reactions are those that generate macrocyclic diterpenes such as casbene and taxadiene (Dueber et al., 1978;Koepp et al., 1995). The second mechanism includes the biosynthesis of a cyclic diphosphate intermediate via class II terpene synthases, the final product being achieved through the class I terpene synthase.
Labdane-type diterpenes represent a distinct class of terpenoids with a characteristic basic bicyclic skeleton connected to an additional six-carbon chain (cyclic or acyclic) that may or may not contains an oxygen atom. Initial data on labdane-type diterpene biosynthesis came from studies on the biosynthesis of ent-kaurene, the diterpenoid precursor of gibberellins, and other labdane-type diterpenes that do not contain oxygen in their skeleton. Ent-kaurene biosynthesis involves a two-step reaction, first the cyclization of GGDP to ent-copalyl diphosphate (ent-CPP) by copalyl diphospate synthase (CPS), and then its conversion to ent-kaurene by ent-kaurene synthase (KS). Copalyl diphosphate synthase performs a protonation-initiated cyclization (class II) while ent-kaurene synthase performs an ionization-initiated cyclization of CPP (class I) (Sakamoto et al., 2004).
The biosynthesis of oxygen-containing labdane-type diterpenes was only recently unraveled. First, it was shown that protein extracts from Nicotiana glutinosa and N. tabacum trichomes that contain labdane-type diterpenes such as abienol, labdenediol, and sclareol, could be converted to all the above oxygen-containing diterpenes in vitro with externally supplied GGDP (Guo et al., 1994;Guo and Wagner, 1995). This observation led to the hypothesis that their synthesis involved a copal-8-ol diphosphate intermediate, the synthesis of which is initiated by protonation of the terminal double bond of GGDP, and the formation of a bicyclic carbocation followed by capture of a hydroxyl anion (Guo and Wagner, 1995). Indeed, it was later shown that this type of enzyme exists.

PHENYLPROPANOIDS
Phenylpropanoid compounds are abundant in the plant kingdom, either providing plants with a valuable defensive arsenal against pathogens, herbivores, and environmental stressors or facilitating the plants reproductive machinery. In addition, these molecules have important applications in the fragnance industry and in medicine. The precursor of all plant phenylpropanoids is trans-cinnamic acid, derived from the amino acid phenylalanine, the first enzymatic step being catalyzed by phenylalanine ammonia-lyase (PAL) leading to the synthesis of p-coumaroyl-CoA, the substrate of more complex aromatic phenypropanoids (Vogt, 2010). Numerous chemicals are classified as plant phenolic secondary metabolites, which are further categorized within several distinct groups based on their basic skeleton as found in simple phenols (C 6 ). The more complex phenylpropanoid compounds include catechol (C 6 )n, phenolic acids (C 6 -C 1 ), phenylacetic acids (C 6 -C 2 ), coumarins (C 6 -C 3 ), lignans (C 6 -C 3 ) 2 , lignins (C 6 -C 3 )n, naphthoquinones (C 6 -C 4 ), xanthones (C 6 -C 1 -C 6 ), stilbenes (C 6 -C 2 -C 6 ), flavonoids (anthocyanins, isoflavonoids, flavones, flavanes, proanthocyanidins, etc.) (C 6 -C 3 -C 6 ) and tannins (C 6 -C 3 -C 6 )n. The biosynthetic pathways of several groups belonging to the very diverse phenylpropanoid compounds have been studied in several model plants, including Arabidopsis thaliana, Meditago truncatula, Nicotiana benthamiana, Oryza sativa (Vogt, 2010). In Cistus, phenolic compounds have been the target of many chemical, biological and taxonomic studies, already discussed in earlier sections of this review and summarized in Table S3. On the basis of these studies, a general pathway of the phenylrpopanoid biosynthesis in Cistus can be drawn ( Figure S2). Several genes involved in the plant phenylpropanoid pathway have been isolated from various plant genera, while some important enzymes were further characterized and novel pathways were discovered (Cheynier et al., 2013). However, the biosynthetic pathway leading to the production of phenylpropanoids and phenolic compounds has not been yet studied in Cistus.

GENOMIC ANALYSES AND BIOTECHNOLOGICAL APPROACHES GENOMIC ANALYSES
Cistus plants are very rich sources of secondary metabolites, which make it hard to isolate quality nucleic acids in sufficient amounts. A protocol for the efficient isolation of high quality DNA and RNA from C. creticus subsp. creticus was published (Pateraki and Kanellis, 2004). This paved the way for a series of molecular studies in C. creticus subsp. creticus aiming for the elucidation of the terpenoid biosynthetic pathway. In view of the valuable properties and possible future exploitation of these natural products it was deemed necessary to study their biosynthesis and its regulation at the molecular level. In this direction, sequence based and functional genomic approaches were initiated. Initial work characterized the expression of genes coding for CcHMGR in the MVA pathway, CcDXS and CcDXR in the FIGURE 2 | Proposed pathway to labdane-type diterpenes predominant in C. creticus resin. A protonation-initiated cyclization catalyzed by CcCLS converts GGDP to the stable bicyclic intermediate copal-8-ol diphosphate. A second ionization-initiated cyclization of copal-8-ol diphosphate results in the formation of manoyl oxide isomers, while labd-13-en-8α,15-diol could be formed either by phosphatase activity or type A diterpene synthase activity. Similarly a different phosphorylated intermediate, labd-7,13-dien-15-yl diphosphate is hypothesized to be converted to labd-7,13-dien-15-ol and then further processed to produce its derivative labd-7,13-dien-15-yl acetate. The acetylated products need the function of novel acetyltransferase(s). Black arrows indicate the biosynthetic steps that involve already characterized enzymes. Question marks indicate pathway steps that have not yet been characterized.
MEP pathway, and followed by the characterization of the two CcGGDPS1 and CcGGDPS2 (Pateraki and Kanellis, 2008). This work suggested that leaf trichomes are very active biosynthetically for terpenoids, and the pathway is regulated at the transcriptional level. Moreover, CcHMGR and CcDXS transcripts (the rate-limiting steps of the isoprenoids' pathways) increase during mechanical wounding or upon treatment with stress hormones such as JA and SA, which possibly reflects an increased need of the plant tissues for the corresponding metabolites (Pateraki and Kanellis, 2010).
The first genes isolated and functionally characterized from C. creticus subsp. creticus were CcGGDPS1 and 2 coding for synthases of geranyl-geranyl diphosphate, the precursor of all diterpenes (Pateraki and Kanellis, 2008). Heterologous expression in Saccharomyces cerevisiae revealed that these full-length cDNAs possessed GGDPS enzyme activity. Gene and protein expression investigations proposed that this enzyme is developmentally and tissue-regulated showing maximum expression in trichomes and smallest leaves (0.5-1.0 cm).
Next, in order to search for putative terpene synthases, an EST library was built using RNA extracted from trichomes isolated from young leaves (Falara et al., 2008). This was decided on the basis of chemical profiling which showed that young leaf trichomes were richer in labdane-type diterpenes compared to mature ones. The subsequent EST analysis that was conducted produced 2022 clones (Falara et al., 2008; http://www. ests-pharm.web.auth.gr/ests.php). Functional annotation of the 2022 expressed sequence tags (ESTs) from the trichome cDNA library, based on homology to A. thaliana genes, showed that 8% of the putative identified sequences belonged to secondary metabolism pathways mainly in flavonoid and terpenoid biosynthesis. Custom DNA microarrays assembled with 1248 individual clones from the cDNA library enabled transcriptome comparisons between trichomes and trichome-free tissues. Verification of the DNA microarrays data by RT-PCR pinpointed a germacrene B synthase (CcGerB) as a trichome specific gene (Falara et al., 2008). Further, the isolation of the promoter of this gene was achieved (Saramourtsi, 2013).
The full-length cDNA of copal-8-ol diphosphate diterpene synthase (CcCLS) was functionally characterized from C. creticus, elucidating the novel first step in the labdane-type diterpenes biosynthetic pathway not only in Cistus but in all angiosperms (Falara et al., 2010, Figure 2). Gene expression analysis revealed that CcCLS is preferentially expressed in trichomes, with higher transcript levels measured in the glandular trichomes of young leaves compared to fully expanded leaves. Interestingly, CcCLS transcript levels increased after mechanical wounding used to simulate herbivore attack. Chemical analyses revealed that labdane-type diterpene production in general followed a similar pattern, with higher concentrations in trichomes of young leaves and increased accumulation upon wounding, indicating that increased diterpene biosynthesis is related to the plant's defense mechanisms. Application of New Generation Sequencing (NGS) in C. creticus trichomes RNA resulted in a total of 385,143 contig sequences (114,239 unigenes) with a mean length of 207 nucleotides. Among those, 2000 unigenes were related to the biosynthesis, transport and catabolism of secondary metabolites. Twenty partial sequences were phylogenetically related to putative diterpene synthases (Papaefthimiou et al., 2013). Among those, two diterpene synthases have already been functionally characterized (see Biosynthetic Pathway for Terpenes Including Labdane-Type Diterpenes).
Another protein isolated and characterized from C. creticus was the key transcriptional regulator TRANSPARENT TESTA GLABRA1 (CcTTG1) (Ioannidi, 2009). In Arabidopsis, TTG1 is involved in trichome differentiation, in the regulation of flavonoids and in seed mucilage production (Walker et al., 1999). The glabrous phenotype of the loss of function A. thaliana ttg1 mutants was restored upon transformation with CcTTG1. The gene was also able to restore the seed coat pigmentation defect of the mutant. The yeast two-hybrid screen resulted in the identification of seven CcTTG1 interactors. Four of them were further analyzed using the yeast-two-hybrid system. Three protein-protein interactions were tested in planta, using a transient expression system in tobacco epidermal cells. The analysis was based on the Bi-molecular Fluorescent Complementation (BiFC) and verified the interaction of CcTTG1 with two Squamosa promoter Binding Proteins (SBP). Also the interactions were detected in the nucleus of the tobacco epidermal cells. This work reported the first protein-protein interaction of the SBP family of transcription factors and a novel interaction of the CcTTG1 protein (Ioannidi, 2009).
Other genetic data available for Cistus is restricted to genes used as molecular markers in taxonomic and phylogenetic studies. Several studies include the isolation and use of partial sequences from a variety of commonly used molecular markers in order to achieve the delimitation of Cistus species. These include the nuclear (ncpGS, ITS) and plastid (trnL-trnF, trnK-matK, trnS-trnG, rbcL) DNA sequences, the trn-F and RPL32-TRNL sequences of cpDNA ) RNA polymerase subunits (Pawluczyk et al., 2012), as well as genetic markers (ISSR-PCR amplification) .

IN VITRO CULTIVATION OF CISTUS
The first in vitro cultivation of Cistus was reported in 1991 (M'Kada et al., 1991) on stem nodal segments for the multiplication of Cistus X purpureus Lam, using MS medium (Murashige and Skoog, 1962) supplemented with cytokinins. Later, a protocol was established for in vitro propagation of Helianthemum almeriense (Cistaceae), using MS with plant growth elicitors for shoot generation (Morte and Honrubia, 1992). Soon after, an improved protocol for in vitro propagation was established with high concentrations of growth elicitors in MS medium leading to successful propagation of six rock-rose species (C. albidus L., C. clusii Dunal, C. ladanifer L., C. laurifolius L., C. psilosepalus L., and C. salviifolius L) (Iriondo et al., 1995). In vitro propagation of C. creticus was successfully established by three separate approaches. In the first approach, a combination of growth elicitors was used on shoots proliferation and on callus regeneration (Pela et al., 2000). In the second approach, shoot formation was obtained 30 days after the first subculture in WPM. The segments were rooted, after supplementing the medium with IBA (0.98-3.94 μM) or NAA (0.1-0.5 μM), and zeatin (0.2-0.5 mg l-1) was used for callus induction (Zygomala et al., 2003). In the third and most recent work, studying the micropropagation of C. creticus (Madesis et al., 2011), rapid proliferation of shoot-tips was achieved using MS, supplemented with growth elicitors, and after 4 weeks, shoots were transferred to MS for rooting or further development. In order to achieve rooting, shoots longer than 1 cm were used and cultured on MS without growth regulators. In vitro proliferation of Cistus has also been studied in C. clusii, with satisfactory results (Ruta and Morone-Fortunato, 2010). In conclusion, methodologies are in place for in vitro cultivation of Cistus plants.