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.

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.

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.

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).

Among volatile organics isolated from leaves of C. albidus collected from southern Catalonia, Spain were docosane, octacosane, and tetracosane (Llusià et al., 2010). Linear hydrocarbons including n-tetradecane and n-hexadecane, were identified exclusively in the pollen and not in other flower parts or leaves of C. albidus growing in Italy in late spring (Maccioni et al., 2007). Only tricosane was detected in the essential oil of C. albidus plants growing in France (Paolini et al., 2008). Several hydrocarbons such as heptacosane, nonacosane, pentacosane, and tricosane were also produced by C. monspeliensis endemic in the South of France, Greece, and Tunisia (Robles and Garzino, 2000; Angelopoulou et al., 2001a; Jemia et al., 2013; Loizzo et al., 2013). Tertadecene, pentacosane, pentadecane, neophytadiene, heptadecene, docosane, heneicosane, and dodecane were all detected in C. parviflorus (Angelopoulou et al., 2001b), while neophytadiene and pentacosane were identified in several Greek and Tunisian populations of C. salviifolius (Demetzos et al., 2002b; Loizzo et al., 2013).

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, 2002b; Robles and Garzino, 2000; Angelopoulou et al., 2001a; Jemia et al., 2013; Loizzo et al., 2013).

Aliphatic aldehydes including octanal, nonanal, decanal and 6-methyl-5-hepten-2-one were present only in the pollen and not in other flower parts or leaves of C. albidus plants growing in Italy in late spring (Maccioni et al., 2007). Nonanal, decanal, undecanal, and dodecanal were detected in the essential oil of C. albidus plants growing wild in France (Paolini et al., 2008). Nonanal and β-ionone were also detected in C. creticus subsp. eriocephalus leaves. Among the secondary metabolites produced by French, Greek and Tunisian C. monspeliensis plants, the norisoprenoids (E)-β-damascenone and members of the ionone family, used in the fragrance industry, as well as several other carbonylic compounds were identified (Robles and Garzino, 2000; Angelopoulou et al., 2001a, 2002; Demetzos et al., 2002b; Jemia et al., 2013; Loizzo et al., 2013).

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 H₂O₂ 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 H₂O₂ 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 (Munné-Bosch et al., 2009).

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 cis-clerodane 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).

Labdane-type diterpenes mainly represented by ent-13-epi-manoyl oxide, manoyl oxide and its isomers were found in significant concentrations in hexane extracts of leaves (16.6%) and fruits (25%) of C. monspeliensis growing in Crete, Greece (Angelopoulou et al., 2001a). Antimicrobial and antifungal activities of C. creticus subsp. creticus and subsp. eriocephalus have been evaluated (Chinou et al., 1994; Demetzos et al., 1995, 1997; Anastasaki et al., 1999; Bouamama et al., 1999; Hutschenreuther et al., 2010). The essential oil extracted from leaves of C. creticus subsp. eriocephalus, which has manoyl oxide and 13-epi-manoyl oxide as its main constituents, exhibited a rather weak activity against E. coli and P. aeruginosa, moderate against Candita albicans, Micrococcus luteus, and S. epidermidis, and most active against S. aureus and Bacillus subtillis (Demetzos et al., 1995), while the volatile oil fraction had a high in vitro activity against Borrelia burgdorferi sensu stricto (Hutschenreuther et al., 2010). The antimicrobial activity of manoyl oxide extracts from C. creticus subsp. creticus fruits, leaves and resin was found dose-dependent against gram-positive Staphylococci species, inactive against gram-negative bacteria (Anastasaki et al., 1999; Demetzos et al., 1999, 2001, 2002c).

Among several tested labdane-type diterpenes, labd-13-en-8α,15-diol was the only diterpene reported to be active against Candida albicans, while labd-7,13-dien-15-ol did not exhibit any antibacterial or/and antifungal activity (Chinou et al., 1994; Bouamama et al., 2006). Several semisynthetic labdane-type diterpenes from the resin “ladano” of C. creticus were reported for their antimicrobial activity both against gram positive and gram negative bacteria and also against pathogenic fungi with the highest effects exhibited by two chloroethyl carbamidic esters derivatives (Kalpoutzakis et al., 2001). C. ladanifer and C. populifolius aqueous extracts had antibacterial activity against E. coli (Barrajón-Catalán et al., 2010).

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 (Barrajón-Catalán et al., 2010). Strong antimicrobial activity against gram-positive bacteria was also demonstrated for C. ladanifer, and against gram-negative bacteria for C. populifolius (Barrajón-Catalán et al., 2010).

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 (Ehrhardt et al., 2007). 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).

Cistus species rich in phenolic compounds, especially flavonoids, demonstrate significant antioxidant properties. Preparations of C. creticus, C. incanus, C. libanotis, C. salviifolius, C. monspeliensis, C. parviflorus, C. laurifolius, C. ladanifer, and C. populifolius aqueous extracts were able to generate strong antioxidant activities in a dose-dependent manner, using several free radical scavenging methods (Attaguile et al., 2000; Sadhu et al., 2006; Teixeira et al., 2007; Amensour et al., 2010; Barrajón-Catalán et al., 2010; Alsabri et al., 2012; Loizzo et al., 2013). Among all studied species, C. monspeliensis appeared to have the highest antioxidant activity (Attaguile et al., 2000, 2004; Loizzo et al., 2013). Potential protective and antioxidant activity was suggested for the pollen of C. incanus, after evaluating the induction of anti-estrogenic properties and differential expression of genes involved in the apoptosis pathway and chemotaxis of mice fed with rich in pollen bees in Croatia (Sarić et al., 2009).

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 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 (Dimas et al., 2006). Sclareol is activated through a p53-independent mechanism that targets the G₁ phase of the cell cycle and therefore apoptosis of human cancer cells is induced through the activation of caspases (Mahaira et al., 2011).

Among nine labdane-type diterpenes isolated from the resin of C. creticus subsp. creticus, labd-13-en-8α-ol-15-diol was active against 13 of the 14 cell lines, while labd-7,13-dien-15-ol showed activity only in HL60 human promyelocytic leukemia cells (Dimas et al., 1998). Pure ent-13-epi-manoyl oxide and mixtures of manoyl oxide isomers isolated from C. monspeliensis and C. creticus aerial parts of plants from Crete were shown to exert moderate cytotoxic activity (Angelopoulou et al., 2001a; Demetzos et al., 2001). Their activity was evaluated against nine leukemic cell lines, where all tested compounds caused growth inhibition at the highest tested concentration of 10⁻⁴ M (Angelopoulou et al., 2001a). In a previous study, however, ent-13-epi-manoyl-oxide did not show any antiproliferative effect on the tested human leukemia cell lines (Demetzos et al., 1994a). A dose- and time- dependent inhibition of DNA synthesis by ent-3β-hydroxy-13-epi-manoyl oxide, was associated with its conversion to a thiomidazolide derivative (Dimas et al., 1999). Both sclareol and the thiomidazolide derivative induced apoptosis of T-cell leukemic cell lines (Dimas et al., 2001). A number of labdane-type diterpenes isolated from aerial parts of C. creticus in Greece, including sclareol, were tested in vitro for their cytotoxic activity against human rhinopharynx cancer, murine leukemia and human bronchial epidermoid carcinoma cell lines (Chinou et al., 1994). Among the tested diterpenes, labd-13-en-8α-ol-15-diol was the only one unable to exhibit nearly any cytotoxic activity. Labd-7,13-dien-15-ol, 8,13-epoxylabd-14-ene (manoyl oxide), 13-epi-8,13-epoxylabd-14-ene (13-epi-manoyl oxide) caused moderate inhibition against the proliferation of the cell lines, while labd-13-en-8α-ol-15-yl acetate, labd-14-en-8,13-diol (sclareol) and 13-epi-sclareol were highly active (Chinou et al., 1994). Moreover, the cis-clerodane (+)-19-acetoxy-cis-clerodan-3-en-15-oic acid, isolated from leaf extracts, was tested against several human leukemic lines and did not exhibit any activity (Demetzos et al., 2001). However, none of the diterpenes tested in the above-mentioned studies from Cretan C. creticus had any anti-inflammatory activity or any effect on skin repair (Demetzos et al., 2001).

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).

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, dose-dependent spasmolytic (Sánchez de Rojas et al., 1995) and analgesic effects (de Andrés et al., 1999) were demonstrated.

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.

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 4-phosphate (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 the MVA pathway, acetyl-CoA from the citric acid cycle undergoes condensation with another acetyl-CoA to produce acetyloacetyl-CoA via the enzyme acetyl-CoA transferase (AACT). Next, HMG-CoA synthatase catalyzes the condensation of one more molecule of acetyl-CoA with acetyloacetyl-CoA to form 3-hydroxy-3-methyl-gloutaryl-CoA (HMG-CoA). HMG-CoA is then reduced to mevalonate by the HMG-CoA reductase (HMGR) using NADPH as co-factor. Mevalonate kinase (MK) activity yields 5-phospho-mevalonate, which then gets decarboxylated to produce IPP. IPP is then isomerized to DMAPP by the enzyme isopentenyl pyrophosphate isomerase (Flesch and Rohmer, 1988; Rohmer et al., 1993).

The first step of the plastidial pathway involves the condensation of pyruvate with 3-phosphoglycerinaldehyde to produce 1-deoxy-D-xylulose 5-phosphate (DXP) (McGarvey and Croteau, 1995). This reaction is catalyzed by 1-deoxy-5D-xylulose synthase (DXS) that functions as a transacetolase (Lange et al., 1998; Lange and Croteau, 1999; Eisenreich et al., 2001). Then 1-deoxy-D-xylulose 5-phosphate is converted to 2-C-methyl-D-erythritol 4-phosphate (MEP) via the reductase DXR (Takahashi et al., 1998). MEP is then converted to 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECPP) in three sequential steps. MEC is converted to 1-hydroxy-2-methyl-2-(E)- butenyl 4-diphosphate by the enzyme 1-hydroxy-2-methyl-2-(E)- butenyl 4-diphosphate synthase (HDS), which is then reduced to 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate via the enzyme 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR). 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate is finally converted to IPP with the enzyme HMBPP reductase. IPP is then isomerized to DMAPP (Lichtenthaler, 1999).

In both cytosolic and plastidic compartments, IPP and DMAPP are used by prenyltransferases to produce prenyl-diphosphates, 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 labdane-type diterpenes that function as phytoalexins in rice (Oryza sativa) involves ent-CPP or syn-CPP as intermediates (Otomo et al., 2004; Prisic et al., 2004; Xu et al., 2004). These phosphorylated intermediates are then further cyclized to the final diterpene products namely oryzalexins, momilactones, and phytocassanes (Nemoto et al., 2004; Otomo et al., 2004; Wilderman et al., 2004; Kanno et al., 2006). All angiosperm terpene synthases may have evolved from the gymnosperm group of bifunctional class II/I terpene synthases producing labdane-related diterpenes. Characteristic examples are: the abietadiene synthase from Abies grandis (Vogel et al., 1996), the levopimaradiene synthase from Ginkgo biloba (Schepmann et al., 2001), and the levopimaradiene/abietadiene from Picea abies (Martin et al., 2004). These bifunctional enzymes catalyze both reactions, first cyclizing GGDP to CPP and then converting CPP to the final tricyclic terpene, that can be further modified by additional enzymes like hydroxylases, methyltranferases, etc. (Ro et al., 2005; Hamberger and Bohlmann, 2006; Ro and Bohlmann, 2006).

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.

C. creticus copal-8-ol diphosphate synthase (CLS) is a type II terpene synthase expressed in the trichomes of C. creticus, which catalyzes the formation of the copal-8-ol diphosphate from GGDP (Falara et al., 2010) (Figure 2). Copal-8-ol diphosphate can then be cyclized to labda-13-en-8α,15-diol, labda-14-en-8,13-diol (sclareol), manoyl-oxide and 13-epi-manoyl-oxide. A similar pathway employing copal-8-ol diphosphate was shown to operate in the trichomes of N. tabacum for the biosynthesis of Z-abienol (Sallaud et al., 2012).

In Salvia sclarea, a class I diTPS (SsSS) has been characterized that transforms the copal-8-ol diphosphate intermediate into sclareol (Caniard et al., 2012; Schalk et al., 2012). In Coleus forskohlii, CfTPS2 catalyzes the synthesis of copal-8-ol diphosphate, which is then utilized by CfTPS3 to its stereospecifically form (13R)-manoyl oxide, the precursor of the highly complex labdene diterpene forskolin (Pateraki et al., 2014). A different phosphorylated intermediate, labd-7,13-dien-15-yl diphosphate, is hypothetically converted to labd-7,13-dien-15-ol and then further processed to produce its derivative labd-7,13-dien-15-yl acetate (Figure 2). In fact, we have recently functionally characterized in E. coli and in yeast a gene from C. creticus that converts GGDP to labd-7,13-dien-15-yl diphosphate (Papaefthimiou et al., 2013). Taken together with the function of CcCLS, we suggest that for each labdane-type diterpene class in Cistus, a unique class II synthase may be required. Recently, a bifunctional labdane-type diterpene synthase producing labd-7,13-dien-15-ol has been characterized from the gymnosperm Selaginella moelendorfii (Mafu et al., 2011).

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 (C₆). The more complex phenylpropanoid compounds include catechol (C₆)n, phenolic acids (C₆-C₁), phenylacetic acids (C₆-C₂), coumarins (C₆-C₃), lignans (C₆-C₃)₂, lignins (C₆-C₃)n, naphthoquinones (C₆-C₄), xanthones (C₆-C₁-C₆), stilbenes (C₆-C₂-C₆), flavonoids (anthocyanins, isoflavonoids, flavones, flavanes, proanthocyanidins, etc.) (C₆-C₃-C₆) and tannins (C₆-C₃-C₆)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.

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 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 (Falchi et al., 2009) RNA polymerase subunits (Pawluczyk et al., 2012), as well as genetic markers (ISSR—PCR amplification) (Paolini et al., 2009).

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.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.