GLANDULAR TRICHOMES

Distribution of Volatile-Containing Glandular Trichomes in the Plant Kingdom

Structures that protrude from the epidermis layer of plants are often referred to as “hairs,” or trichomes, and they appear to perform a variety of functions (Schilmiller et al., 2008; Tissier, 2012; Lange, 2015). A subset of trichomes are defined as “glandular,” because they contain one or more cells that actively synthesize and store specialized compounds, some of which may be volatile (Tissier, 2012). Glandular trichomes come in many shapes and levels of complexity even in the same plant (Tissier, 2012; Lange, 2015), from a single cell to more elaborate structures. Their biochemical repertoire is also extremely diverse (Schilmiller et al., 2008), and includes different classes of compounds, some of w'hich are volatile and some are not. Some glandular trichomes continuously secret their compounds, and others store them extracellularly and these compounds are released into the open only w'hen the trichomes are physically impacted. However, w'ith the exception of some glandular trichomes in ferns that produce nonvolatile farinose waxes, reports of volatile compounds detected in glandular trichomes are restricted to flowering plants so far, and mostly to eudicotyledenous plants at that (Tissier, 2012).

Moreover, even among the eudicots, the vast majority of reports of volatile-producing glandular trichomes come from just a few' families, including the Lamiaceae, Solanaceae, Asteraceae, and Cannabaceae (Tissier, 2012) (Figure 13.1). And the vast majority of these volatile chemicals are terpenoids, with benzenoids constituting a second but much smaller class, and a smattering of fatty acid derivatives, amino acid derivatives, and polyketides rounding up the rest. In the following sections, the occurrence of volatiles in glandular trichomes will be described using a phylogenetic reference frame.

FIGURE 13.1 Examples of volatiles in glandular trichomes. (a) Lamiaceae, (b) Solanaceae, (c) Asteraceae, and (d) Cannabaceae.

13.2.1.1 Lamiaceae

This family, with approximately 7,000 species, contains many well known scented plants, such as basil, mint, rosemary, sage, oregano, thyme, lavender, and perilla. There are many reports of the occurrence of various types of glandular trichomes in Lamiaceae species. However, most reports on the presence of volatiles in trichomes in this family, as indeed in many other families described below, follow indirect routes to placing the volatiles in the trichomes. Typically, the presence of trichomes on specific organs is first noted, sometimes followed by histochemical staining of the trichomes using chemical stains that are presumed to identify specific classes of chemicals. Then, volatiles are extracted from the whole organ (e.g., leaf, stem, or flower) and chemically analyzed by gas chromatography-mass spectrometry (GC-MS). The volatiles identified are then assumed to come from the trichomes. This is probably a good assumption for volatiles found in large amounts that, because of their toxicity, are unlikely to be present in any structures other than glandular trichomes (where they are typically stored in the space between the outer membrane of the cell and the cuticle). However, minor volatile compounds identified in such investigations are just as likely to come from cells other than those making up the glandular trichomes. This potential shortcoming being noted, it is nevertheless becoming more common to isolate trichomes and directly analyze their volatiles by GC-MS, or to directly sample volatiles from trichomes with micro-sampling techniques without first separating the trichomes from the rest of the plant (Gang et al., 2001; Falara et ah, 2008; McDowell et ah, 2011).

With these caveats, the wealth of data on trichome volatiles in Lamiaceae points to the dominance of terpenes in these structures. In some species, volatile monoterpenes predominate, while in others, sesquiterpenes constitute the majority. For example, mint (Mentha spicata) trichomes contain high levels of the commercially important monoterpene menthol, while the trichomes of Pogostemon cablin produce mostly sesquiterpenes, including patchoulol, a sesquiterpene alcohol highly valued for perfume (Figure 13.1) (Maffei et ah, 1986; Deguerry et ah, 2006). In addition to the “standard” monoterpenes and sesquiterpenes that occur in the glandular trichomes throughout this plant family, some species in the Lavandula genus also make some unusual, or “irregular,” monoterpenes in their trichomes (Figure 13.1). These irregular monoterpenes include lavandulol, which is derived from the condensation of two DMAPP molecules, and lavandulyl acetate (Demissie et ah, 2013).

Species in Ocimum, another genus in the Lamiaceae family, are noted for the presence of phenylpropenes in their trichomes, such as eugenol, methyleugenol, chavichol, and methylchavichol, in addition to mono- and sesquiterpenes (Gang et ah, 2001, 2002; Raina et ah, 2018). In some cultivated basil (O. basilicum) lines that are highly bred, phenylpropenes could constitute up to 75% of total volatiles (Iijima et ah, 2004). It should be borne in mind that Lamiaceae trichomes often contain high amounts of nonvolatile compounds as well, much of it consisting of chemicals that belong to the phenylpropanoid class (from which the phenylpropones are derived), such as rosmarinic acid, caffeic acid, etc. (Gang et ah, 2002).

Volatiles belonging to other classes of chemicals that have sometimes been reported for Lamiaceae species include straight-chain alkanes, aldehydes, and alcohols, but their low concentrations makes their assignment to the trichome less secure. However, Maggi et ah (2010) reported that the plant known as bastard balm (Melittis melissophyllum subsp. melissophyllum), a perennial herb growing in Italian woodlands, contains high levels of l-octen-3-ol, up to 70% of the total essential oil (most of the rest are terpenoids). While a direct proof that this compound is present in the trichomes was not presented, the authors did show that the aerial surfaces of this plant are covered by several types of glandular trichomes, making it likely that l-octen-3-ol is indeed present in the trichomes.

13.2.1.2 Solanaceae

The Solanaceae (nightshade) family, with about 2,700 species, is considerably smaller than the Lamiaceae but appears to contain as much volatile chemical diversity in the various types of glandular trichomes that its species possess. Within the family, investigations of the chemistry of glandular trichomes have so far been essentially restricted to the large Solanum genus, which includes the cultivated tomato (S. lycopersicum), and the much smaller Nicotinana genus, which includes the cultivated tobacco (N. tabacum),

The recently enlarged genus Solanum now contains about half of the species in the family, but most of the work on volatiles has concentrated on the cultivated tomato and closely related species. Both cultivated and wild tomato plants contain multiple types of glandular trichomes on their leaves, stems, developing fruits, and sometimes on the flowers (Figure 13.2). The glandular trichomes with a single biosynthetically active cell at the top of the stock (designated as type 1 and type 4, depending on the length of the stock) appear to produce mostly toxic, sticky, nonvolatile compounds such as acylated sugars (Schilmiller et al., 2010; McDowell et al., 2011). On the other

FIGURE 13.2 Examples of plant glandular trichome diversity, (a) Scanning electron microscopy (SEM) image of a young wild tomato (Solatium habrochaites, Solanaceae, accession LA2409) leaf. This picture illustrates the complexity of the leaf trichome landscape. At least five trichome types, numbered according to Luckwill (1943) can be observed: Type 1, tall glandular trichomes with a single secretory cell; Type 2, tall non-glandular trichomes; Type 3, short hooked non-glandular trichome; Type 6, glandular trichomes with four head cells; Type 7, short glandular trichomes. (b) Detail of a tomato leaf (Solatium lycopersicum LA 4024) showing Type 6 and Type 3 trichomes. (c) Detail of a wild tomato leaf (S. habrochaites LA 1777) showing Type 4, Type 6 and Type 7 trichomes. In S. habrochaites, the four glandular cells of Type 6 trichomes are enveloped in a peri-cellular cuticle, whereas in the cultivated tomato the four glandular cells can be distinctly seen (see b). (d) Capitate trichome of Nicotiana sylvestris (Solanaceae), similar to Type 4 trichomes of tomato. A droplet of diterpenoid-rich exudate can be seen on the side of the glandular head (white arrow), (e) Scanning electron microscopy (SEM) image of a floret from Tanacetum parthenium (Asteraceae) showing files of glandular trichomes. (f) A close-up view of the same type of glandular trichome in light microscopy showing the large subcuticular cavity where hydrophobic volatile compounds are stored, similar to the peltate trichomes of the Lamiaceae. (g) Scanning electron microscopy (SEM) image of a Salvia officinalis (Lamiaceae) leaf, showing peltate trichomes in a network of hairs, (h, i) Detailed view of a peltate trichome of S. officinalis, showing the envelope delimiting the subcuticular storage space (h), and the plate of eight glandular cells (i). (Reproduced, with permission, from Tissier, A., Plant J., 70, 51-68, 2012.) hand, glandular trichome type 6, which consists of four cells forming a sphere on top of a long stock, appears to specialize in producing volatiles. The type 6 glandular trichomes of various Solatium species, such as 5. lycopersicum, S. pimpinellifolium, S. pennellii, and S. habroichaites, produce and store a large repertoire of monoterpenes and sesquiterpenes (Sallaud et al., 2009; Falara et al., 2011; McDowell et al., 2011). Even within the same species, type 6 glands found on one organ, such as leaves, may have a somewhat different composition of terpenoid volatiles than the type 6 glands found on stems (Falara et al., 2011). In some cases, glandular terpenes are extensively oxidized and are turned into less-volatile, sticky acids, as happens to a large portion of the sesquiterpene berga- motene and santalene in the type 6 glands of 5. habrochaites (Sallaud et al., 2009).

Solarium type 6 glands exhibit a unique biochemical innovation in how they synthesize some of their mono- and sesquiterpenes. While some Solarium terpene synthases (TPSs) present in type 6 glands use the canonical substrates (GPP for monoterpene synthases and c,e-FPP for sesquiterpene synthases), others use neryl diphosphate (NPP), the m-isomer of GPP, or z,z-FPP, respectively (Schilmiller et ah, 2009, Gonzales-Vigil et ah, 2012). The m-prenyldiphosphate substrates are produced in the plastids only. Not only are the majority of monoterpenes in type 6 glands derived from NPP rather than GPP (Schilmiller et ah, 2009; Gonzales-Vigil et ah, 2012), in certain 5. hab- rochaites accession the majority of the sesquiterpenes produced in these glands are made from or Z.Z-FPP rather than e,e-FPP, and, furthermore, are made in the plastids of the trichome rather than in the cytosol, where sesquiterpenes are usually synthesized (Sallaud et ah, 2009). Interestingly, there is no evidence that the use of m-prenyldiphosphate substrates allows for different type of monoterepenes or sequiterpenes to be made, as many of the same monoterpenes and sesquiterpenes produced from m-prenyldiphosphates (Figure 13.1) are produced in other plant species from the corresponding /ran.v-prenyldiphosphates. Thus, the advantage for the evolution of this alternative pathway for making mono- and sesquiterpenes is not clear.

Another innovation found in type 6 glands in Solarium is the synthesis of a class of volatiles called methylketones. Several S. American tomato species synthesize methylketones, mostly 2-undecanone and 2-tridecanone, but the concentrations of these compounds are particularly high in several accessions of S. habrochaites (Antonious, 2001; Fridman et ah, 2005). In these accessions, the synthesis of terpenes in these glands is correspondingly low (Ben-Israeh, 2009). The biosynthesis of methylketones in Solatium has been shown to involve two enzymes, the first being and esterase that interferes with fatty acid biosynthesis by hydrolyzing the carbon-sulfur bond in the 3-ketoacyl-ACP intermediate to produce a Cll or a C13 3-ketoacid, and a second enzyme that brings about decarboxylation (Fridman et ah, 2005; Ben-Israeh, 2009; Yu et ah, 2010). 2-Undecanone and 2-tridecanone are somewhat volatile, but their role is believed to be in defense against insect herbivores, to which these methylketones are toxic (Williams et ah, 1980). Recently, 2-undecanone was detected in the sticky exudate of S. sarrachoides, an African species in the Solarium nigrum complex (Murungi et ah, 2016).

Volatiles are important flavor compounds in smoking tobacco. Many of them are formed during the leaf-curing process (which often involves heat as well as catabolism by microbial and plant enzymes) and through pyrolysis during smoking, and so they do not constitute plant volatiles. However, extensive research on the origin of tobacco flavor has shown that many of these volatile/ flavor compounds are derived from diterpenes produced in the glandular trichomes on leaves of N. tabacum, and these diterpenes are nominally volatile by themselves. Foremost among them is the labdane-type diterpene z-abienol (Sallaud et ah, 2012) (Figure 13.1), and another group of semi-volatile diterpenes produced by these glands are the macrocyclic cembratrien-diols (Guo and Wagner, 1995). Some tobacco varieties also produce cembratrien-ols (the precursors of the cembratrien-diols) which are more volatile, and actually appear to be more toxic to aphids (Wang et ah, 2001).

13.2.1.3 Asteraceae

With more than 30,000 species, the Asteraceae is one of the two largest families in the angiosperms (the other is Orchidaceae). Many Asteraceae species have been reported to have glandular trichomes (Figure 13.2). In many cases, sesquiterpene lactones (Figure 13.1), which are only minimally volatiles, have been found in these trichomes (Lopes et ah, 2013; Ramirez et ah, 2012, 2013). However, when examined more closely, these glands are also found to hold more volatile terpenes. For example, the glands of Artemesia annua, in addition to the malaria drug sesquiterpene lactone artemisinin, also contain a large number of volatile mono- and sesquiterpenes, with the monoterpenes pinene or camphor dominating, depending on the plant accessions examined (Tellez et ah, 1999; Yadav et ah, 2014). Stevia rebaudiana glandular trichomes, the source of the diterpene glycosides that serve as natural sweeteners, also synthesize and store a variety of volatile mono- and sesquiterpenes (Woelwer-Rieck et ah, 2014). The trichomes of the Mexican medicinal plant Montanoa tomentosa contain the two diterpene acids which are believed to be responsible for its medicinal properties (including inducting childbirth), but also a number of monoterpenes and sesquiterpenes, with valencene, p-eudesmol (Figure 13.1), and geranyl acetate being the most abundant (Robles-Zepeda et al., 2009). And Piazza et al. (2018) report that the capitate glands of the Argentinian species Flourensia campestris contain a large number of volatile mono- and sesquiterpenes, as well as small amounts of two polyketide acetopenones.

13.2.1.4 Cannabaceae

Two species in particular have been extensively studied in this family in respect to volatiles in glands, Cannabis saliva (the marijuana plant) and Hamulus lupulus (hop). Both are diecious plants, and the bracts of the female inflorescences in both species are particularly covered with a dense array of glandular trichomes. In C. saliva, these glands contain the psychoactive chemical tetrahydrocannabinol (THC), which is a nonvolatile compound made from a backbone of a prenyl- ated (geranylated) polyketide. However, in addition to THC these glands also contain high amounts of volatile monoterpenes (mostly a-pinene, р-pinene, myrcene, limonene, p-ocimene, and terpin- olene) and sesquiterpenes (mostly р-caryophyllene) (Booth et al., 2017). In the glands of the closely related hop plants, these glands contain high levels of the monoterpene myrcene and the sesquiterpenes р-caryophyllene and a-humulene (Wang et al., 2008). Hop glands also contain prenylated (and methylated) polyketides such as humulone and xanthohumol (Figure 13.1) which are somewhat volatile, a property that allows them to contribute to beer flavor when added during the beer making process (Nagel et al., 2008).