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Keywords:

  • glandular trichomes;
  • transcriptomics;
  • proteomics;
  • development;
  • modeling

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Techniques for Collecting and Purifying Trichomes
  5. Glandular Trichomes: Secreting and Producing Glands
  6. Transcriptomics and Proteomics of Glandular Trichomes
  7. Metabolic Pathway Discovery
  8. Metabolic Profiling of Glandular Trichomes
  9. Glandular Trichome Modeling
  10. Future Directions
  11. Acknowledgements
  12. References

Glandular trichomes cover the surface of many plant species. They exhibit tremendous diversity, be it in their shape or the compounds they secrete. This diversity is expressed between species but also within species or even individual plants. The industrial uses of some trichome secretions and their potential as a defense barrier, for example against arthropod pests, has spurred research into the biosynthesis pathways that lead to these specialized metabolites. Because complete biosynthesis pathways take place in the secretory cells, the establishment of trichome-specific expressed sequence tag libraries has greatly accelerated their elucidation. Glandular trichomes also have an important metabolic capacity and may be considered as true cell factories. To fully exploit the potential of glandular trichomes as breeding or engineering objects, several research areas will have to be further investigated, such as development, patterning, metabolic fluxes and transcription regulation. The purpose of this review is to provide an update on the methods and technologies which have been used to investigate glandular trichomes and to propose new avenues of research to deepen our understanding of these specialized structures.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Techniques for Collecting and Purifying Trichomes
  5. Glandular Trichomes: Secreting and Producing Glands
  6. Transcriptomics and Proteomics of Glandular Trichomes
  7. Metabolic Pathway Discovery
  8. Metabolic Profiling of Glandular Trichomes
  9. Glandular Trichome Modeling
  10. Future Directions
  11. Acknowledgements
  12. References

When one looks at plant surfaces with a scanning electron microscope, a picture of bewildering diversity of shapes and structures comes to view. The first level of complexity lies at the very surface of the cells where the outermost layer of the cuticle, i.e. the epicuticle, produces various elements such as wax crystals (Samuels et al., 2008). This is in the sub-micrometer range and is not within the scope of this review. When one zooms out, a surprising micro-landscape of tree- and bush-like structures is encountered in many species. These protuberances, which range in size from a few microns to several centimeters, are called trichomes. Their diversity is almost as great as the number of species on which they are encountered, as they can be single-celled or multicellular, curved or straight, non-secretory or glandular, and with many more descriptive criteria possible (Werker, 2000). A glossary of terms depicting trichomes with over 700 entries was even compiled, giving us an idea of the range of shapes that may be encountered (Payne, 1978). There is, however, one criterion which is commonly used to classify trichomes, namely whether they are glandular or not. Glandular trichomes also encompass a great variety of shapes and structures, but have in common the presence of metabolically active cells with, for many of them, the capacity to secrete or store large quantities of specialized metabolites. The distribution of glandular trichomes in the plant kingdom suggests a fairly ancient evolution. Peltate trichomes can be observed in fossilized leaf material from seed ferns such as Barthelopteris germarii of the late Paleozoic (Krings and Kerp, 1998) and multicellular glandular trichomes from the seed fern Blanzyopteris praedentata of the late Carboniferous (Krings et al., 2003). The latter are compared with the explosive glandular trichomes of extant angiosperms such as Sicana odorifera (Cucurbitaceae), suggesting that these fern glandular trichomes may have been part of a defensive strategy against arthropod pests (Krings et al., 2002, 2003). Studies on living ferns, in particular the Pteridaceae, also indicate the existence of glandular trichomes which are involved in the production of a farinose wax composed mostly of flavonoid aglycones (Wollenweber et al., 1998; reviewed in Wollenweber and Schneider, 2000). No record of glandular trichomes from bryophytes could be found, suggesting that glandular trichomes are an invention of vascular plants. Although hairs are found on twigs of gymnosperms, such as Picea sp. where they are used as taxonomic markers (Fernald, 1950; cited in Johnson, 1975), glandular trichomes per se are rare and were reported only in juvenile leaves of Pinus cembra and Pinus lambertiana (Napp-Zinn, 1966). In contrast, gymnosperms are endowed with another type of secretory cell, namely those of the resin ducts, which are located inside the plant body (Fahn, 1988). Glandular trichomes occur very frequently in angiosperms where they can be found in many plant families (Fahn, 1988; Werker, 2000; Wagner et al., 2004). However, a comprehensive survey of the occurrence and types of glandular trichomes across the angiosperm clade is missing and would certainly provide more insight into the evolutionary patterns of these structures. Species of some angiosperm dicotyledonous families such as the Lamiaceae, Solanaceae, Asteraceae and Cannabaceae are particularly rich in glandular trichomes. Monocotyledons are less known for their glandular trichomes, but there are well-studied cases of the oil-producing trichomes which occur in flowers of species from the Liliaceae and the Orchidaceae and play critical roles in pollination by specialized bees (Buchmann, 1987). Fahn (1988, 2002) has summarized and reviewed the current hypotheses on the evolution of plant secretory structures and of glandular trichomes in particular. It is suggested that in the course of evolution, the secretory cells were originally located in the mesophyll and subsequently migrated either to internal vascular tissues or to the outside to generate glandular trichomes. According to this theory, glandular trichomes would represent the most recent and advanced type of secretory tissue (Fahn, 1988). There is, however, no molecular support for this hypothesis, and the sheer diversity of structures and shapes of glandular trichomes would indicate that they may not be the result of a single evolutionary event. On the contrary, a study on basal angiosperms suggests that trichomes may have evolved from stomata several times independently (Carpenter, 2006). Even within a single genus, Sisyrinchium (Iridaceae), phylogenetic analysis indicates that glandular trichomes have evolved three times independently (Chauveau et al., 2011). In addition, in some cases, morphological analogies between multicellular non-glandular and glandular trichomes have suggested that glandular trichomes have evolved from non-glandular trichomes through the differentiation of apical cells into secretory cells (Uphof, 1962; Fahn and Shimony, 1977). One such example is provided by leaf trichomes of tomato species (Solanum lycopersicum and Solanum habrochaites) where the capitate trichomes of Type 1 only differ from the long non-glandular trichomes of Type 3 by the presence of a single secretory cell at the tip (Luckwill, 1943) (see Figure 1A).

image

Figure 1.  A sample of plant glandular trichome diversity. (A) Scanning electron microscopy (SEM) image of a young wild tomato (Solanum habrochaites, 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 (Solanum 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, 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 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 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).

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Glandular trichomes can also be classified according to the type of compounds they produce: hydrophilic, lipophilic, proteins, poly- or monosaccharides. Further, they may be classified according to the volatility of the compounds they produce. Volatile compounds such as mono- and sesquiterpenes, phenylpropenes or methylketones, if not retained one way or another would simply diffuse into the headspace. Some glandular trichomes, like the peltate trichomes of the Lamiaceae (mint family) are particularly well adapted to retain and store volatile compounds [in mint (Mentha sp.) or sage (Salvia officinalis) (see Figure 1G–I) for example]; a subcuticular storage cavity which results from the separation of the cuticle from the cell wall lies above the plate of eight glandular cells. Upon rupture of the cuticle, which may occur when an insect treads upon the trichome, or when the leaves are crushed, the volatile compounds of this storage space are released and can then diffuse into the headspace. Similar storage spaces are also found in glandular trichomes of Asteraceae, for example Tanacetum parthenium (see Figure 1E,F). On the contrary, capitate trichomes, such as those of tobacco, synthesize compounds, which are not volatile or are poorly volatile and are directly exuded onto the surface of the trichome (Figure 1D). The droplets of exudate can be seen coming out of the glandular cells at the tip of the trichome and dripping down the trichome stalk. In some cases, as in tobacco or in the wild tomato Solanum pennellii, the exudate is highly sticky and sets a trap for insects or small arthropods, which can become glued to the leaf and die (Fobes et al., 1985). In most species however, several types of trichomes, non-glandular and glandular, capitate and peltate, coexist. The complexity of the trichome landscape is illustrated by a scanning electron microscopy image of a young leaf of a wild species of tomato (Solanum habrochaites ssp. glabratum) (Figure 1A). The diversity of trichomes is mirrored by the metabolic diversity of compounds they produce. As an example of this diversity, some compounds produced by tomato glandular trichomes, which will be referred to later in this review, are presented in Figure 2.

image

Figure 2.  Examples of metabolites found in glandular trichomes of various tomato species. (+)-α-santalene, (+)-endo-β-bergamotene, 7-epizingiberene and their respective carboxylic acids are found in Type 6 trichomes of Solanum habrochaites. 2-Undecanone is found in Type 6 trichomes of S. habrochaites ssp glabratum. Glucose esters of short chain fatty acids are produced by Type 4 trichomes of Solanum pennellii. The tetra-methylated myricetin is found preferentially in Type 4 trichomes of S. habrochaites. β-Phellandrene is produced in Type 6 trichomes of S. lycopersicum.

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The secretions of glandular trichomes have been exploited by humans for a number of uses and are only illustrated by a couple of examples here. For a more detailed presentation of this topic, readers are referred to a recent review (Schilmiller et al., 2008). Probably one of the most ancient uses stems from the highly fragrant and aromatic properties of some of those secretions. Collected by distillation they are referred to as essential oils. The plant family which contributes the most essential oils is the Lamiaceae, with popular species such as mint, basil, lavender, oregano or thyme; clary sage (Salvia sclarea), is grown for its essential oil, but particularly for its concrete, which contains a high content (up to 50%) of sclareol. Sclareol, a labdanoid diterpene, is produced in capitate trichomes of the inflorescences and is used as a synthesis precursor of fixative agents such as Ambrox or Ambroxane, which are highly prized ingredients in the fragrance and detergent industry (Moulines et al., 2001, 2004; Barrero et al., 2004; Frija et al., 2011). Secretions of glandular trichomes have been used for their medicinal properties, and in some cases active ingredients have been marketed as drugs. Artemisinin is a sesquiterpene lactone produced in glandular trichomes of Artemisia annua and used for the treatment of malaria as an alternative to quinine drugs, which face increasing resistance from emerging strains of the malarial parasite (Covello, 2008; Weathers et al., 2011). Pharmaceutical companies have developed derivatives of artemisinin (artemeter, artesunate) which are now widely marketed (Shanks, 2006).

In this review, I will provide an update on the methods and techniques which have been used to study glandular trichomes, and how this has allowed the elucidation of novel pathways for specialized metabolites. However, the interest in glandular trichomes also lies beyond the characterization of biochemical pathways, namely in the understanding of how these cell factories develop and function in order to be in a position to fully exploit their potential. Research avenues on how to achieve this goal are proposed, with, among others, the adoption of model species, comprehensive metabolic profiling integrating primary and specialized metabolism, detailed developmental studies and the functional analysis of genes by forward or reverse screens.

Techniques for Collecting and Purifying Trichomes

  1. Top of page
  2. Summary
  3. Introduction
  4. Techniques for Collecting and Purifying Trichomes
  5. Glandular Trichomes: Secreting and Producing Glands
  6. Transcriptomics and Proteomics of Glandular Trichomes
  7. Metabolic Pathway Discovery
  8. Metabolic Profiling of Glandular Trichomes
  9. Glandular Trichome Modeling
  10. Future Directions
  11. Acknowledgements
  12. References

Different methods have been tested and developed to specifically harvest plant trichomes, and in particular glandular trichomes. The peculiarities of different types of glandular trichomes have necessitated the development of adapted techniques which, while preserving the integrity of the glandular cells, allowed a sufficiently pure preparation. The most basic technique relies on abrasion of the leaf surface with a brush or a microscope glass slide. This was applied to harvest mint (Croteau and Winters, 1982) and tobacco trichomes (Keene and Wagner, 1985) and provided material that demonstrated that enzymes for the biosynthesis of compounds which are secreted by trichomes are also present in trichomes (see below). Later, the techniques were somewhat refined with the use of glass beads to detach glands followed by either a density gradient, e.g. in Percoll, or a succession of meshes of appropriate sizes to purify trichomes (Gershenzon et al., 1987, 1992). This glass bead–mesh technique has turned out to be the most successful and it has been applied to numerous other species, including basil (Gang et al., 2001), tomato (van Der Hoeven et al., 2000; Fridman et al., 2005; Slocombe et al., 2008; Schilmiller et al., 2010b), hops (Nagel et al., 2008; Wang et al., 2008), Artemisia annua (Teoh et al., 2006), and sage (Salvia fruticosa) (Chatzopoulou et al., 2010). Abrasion with powdered dry ice followed by mesh filtration was also used successfully to prepare heads of glandular trichomes from geranium, potato and tomato among others (Yerger et al., 1992). This method presents the advantage that the glandular cells are frozen as they are harvested, thus providing high-quality material for RNA and protein isolation. Glandular trichomes from a number of species, including Cistus creticus, Cistus laurifolius, Cannabis sativa and Nepeta racemosa, were successfully prepared with this method, demonstrating its robustness and reliability (Clark et al., 1997; Hallahan et al., 1998; Sirikantaramas et al., 2005; Falara et al., 2008; Pateraki and Kanellis, 2010). Variations of these techniques were developed, for example in tobacco or Medicago sativa, by using leaves frozen in liquid nitrogen but abrading them with glass beads followed by mesh filtering (Ranger et al., 2004; Amme et al., 2005). Other methods rely on the trapping of glandular trichomes on adhesive tape and release of the trichomes from the tape by water (Yamaura et al., 1992) or the use of double-sided tape stuck to a glass slide (Gopfert et al., 2006). More recently, RNA from single glandular cells of A. annua trichomes could be recovered after laser microdissection. The A. annua glandular trichomes contain several layers of secretory cells, and the expression of artemisinin pathway genes could be shown to be preferentially located in the apical cells (Olsson et al., 2009). However, the same authors recently published an improved method and demonstrated that genes of the artemisinin pathway are expressed in all cells (apical and sub-apical) (Olofsson et al., 2012). Furthermore, specific expression in all secretory cells of the Artemisia glandular trichomes could be shown in transgenic plants carrying AaAS-promoter:GUS and CYP71AV1-promoter:GUS fusions (Wang et al., 2011).

Glandular Trichomes: Secreting and Producing Glands

  1. Top of page
  2. Summary
  3. Introduction
  4. Techniques for Collecting and Purifying Trichomes
  5. Glandular Trichomes: Secreting and Producing Glands
  6. Transcriptomics and Proteomics of Glandular Trichomes
  7. Metabolic Pathway Discovery
  8. Metabolic Profiling of Glandular Trichomes
  9. Glandular Trichome Modeling
  10. Future Directions
  11. Acknowledgements
  12. References

Early studies on the biochemistry and physiology of glandular trichomes pointed to the fact that intense metabolic activity takes place in the glandular cells and that they could be the site of biosynthesis of the secreted compounds. This was first observed by ultrastructural and cytochemical studies, for example in peppermint (Amelunxen, 1965; Amelunxen et al., 1969; Lange et al., 2000). The first biochemical evidence came with the feeding of 14C-acetate to tobacco leaf epidermal peels containing trichomes, or whole trichomes collected by ‘shaving’ the stems. 14C was incorporated into cembratrien-diols (CBT-diols, formerly called duvatrien-diols), the major diterpenes secreted by glandular trichomes (Michie and Reid, 1968). An apparently contradictory view was supported by the observation that CBT-diols were found on the whole surface of the epidermis, suggesting that they may not be synthesized exclusively in the trichomes but also in the epidermal cells (Chang and Grunwald, 1980). However, this can be easily attributed to the fact that the secretion from glandular heads, although viscous, is fluid and slowly spreads over the surface of the leaf (Figure 1D). A subsequent labeling study, performed with purified trichome heads and not just a whole trichome fraction, convincingly demonstrated incorporation into CBT-diols (Keene and Wagner, 1985). In retrospect, 14C-acetate was certainly not the best precursor for such feeding studies. Its use was based on the assumption that diterpenes would be synthesized through the mevalonate pathway, which was the only known isoprenoid precursor pathway at the time. It is now well established that the alternative 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway provides isoprenoid precursors in the plastids of higher plants, and that diterpenes, which are synthesized in plastids, are produced with those MEP-derived precursors (Phillips et al., 2008). This pathway starts with pyruvate and glyceraldehyde-3-phosphate, which would be better suited to perform labeling studies. Interestingly, the low level of incorporation of acetate into tobacco diterpenoids indicates either a recycling of acetate into pyruvate through the citric acid cycle or alternatively import of mevalonate-derived precursors into the chloroplast. Further biochemical evidence that tobacco glandular trichomes are the site of biosynthesis of secreted compounds was provided by feeding 14C-sucrose and 14C-glucose. Both sucrose esters and diterpenoids could thus be labeled in detached trichome glands (Kandra and Wagner, 1988).

Another line of evidence for the in situ biosynthesis of trichome-secreted compounds came from studies in spearmint, where limonene hydroxylase and carveol dehydrogenase activities from extracts of glandular trichomes could be demonstrated (Gershenzon et al., 1989). Further, immunolocalization studies in spearmint and peppermint provided an even more refined picture. The limonene synthase and both subunits of the geranyl diphosphate synthase could be localized in the leucoplasts of the glandular cells, while the cytochrome P450 monooxygenase, 4-S-limonene-6-hydroxylase, was detected in the smooth endoplasmic reticulum of spearmint (Turner et al., 1999; Turner and Croteau, 2004). Two other enzymes involved in downstream steps of the pathway, namely (−)-trans-isopiperitenol dehydrogenase and peppermint (−)-pulegone reductase, were detected in the mitochondria and the cytosol, respectively (Turner and Croteau, 2004). This shows that in a single cell type several distinct intracellular compartments are involved in the biosynthesis of the final compounds. This raises the question of intracellular transport of pathway intermediates, and it was proposed that a combination of free diffusion and selective active transport could account for this partitioning (Turner and Croteau, 2004).

The cloning of genes involved in the trichome metabolic pathways allowed the characterization of their promoters in transgenic plants. This was done in tobacco, and nicely showed that their expression is restricted to the glandular cells of the trichomes and therefore highly specific (Wang et al., 2002; Ennajdaoui et al., 2010). In addition to these tobacco promoters, however, there are relatively few trichome-specific promoters known from genes of the endogenous pathways. The only ones described in the literature are the promoter of the linalool synthase from lavender (Biswas et al., 2009), the promoter of the A. annua amorphadiene synthase (AaAS) and that of a WRKY transcription factor which binds to the AaAS promoter (Kim et al., 2008; Ma et al., 2009). For more detailed information on trichome-specific promoters, readers are referred to a recent review on the topic (Tissier, 2012).

Transcriptomics and Proteomics of Glandular Trichomes

  1. Top of page
  2. Summary
  3. Introduction
  4. Techniques for Collecting and Purifying Trichomes
  5. Glandular Trichomes: Secreting and Producing Glands
  6. Transcriptomics and Proteomics of Glandular Trichomes
  7. Metabolic Pathway Discovery
  8. Metabolic Profiling of Glandular Trichomes
  9. Glandular Trichome Modeling
  10. Future Directions
  11. Acknowledgements
  12. References

The advent of genomics and transcriptomics allowed the large-scale sequencing of expressed sequence tags (ESTs) (Adams et al., 1991). The application of EST sequencing to purified trichomes, generated collections of sequences which have proved extremely useful for the identification and characterization of pathway genes (see below). The EST projects from three species (mint, basil and tomato) will be presented in more detail below, whereas the others are listed in Table 1. In total, glandular trichome EST projects were carried out in 16 different species with more than 2 million sequences. In contrast, only a few proteomic studies are reported, with one in basil, two in tobacco and one in tomato (Amme et al., 2005; Xie et al., 2008; Schilmiller et al., 2010b; Van Cutsem et al., 2011). The first trichome EST sequencing project was carried out in mint with 1316 sequences obtained by the Sanger sequencing method (Lange et al., 2000). Although 1316 is a relatively low number of EST sequences, this was enough to identify genes in the MEP and the menthol biosynthesis pathways. Moreover, 25% of the described sequences in this pool were estimated to be involved in essential oil metabolism. Among the most highly expressed genes in the terpenoid pathway were the (−)-limonene-3-hydroxylase (61.1‰), (−)-limonene synthase (29.6‰), and isopiperitone reductase (22.4‰). Genes of primary metabolism were also highly expressed with glycolysis, pentose phosphate and oxidative phosphorylation pathways accounting for 17, 8.8 and 9.6‰ of the ESTs, respectively. Another important group of highly expressed genes were homologs of lipid transfer proteins (LTPs) with 32‰. Although their function in the trichomes is not yet clearly defined, it is assumed that they play a role in the transport of the lipophilic terpenoids to the subcuticular storage cavity of the peltate glands. A similar approach was chosen to discover genes involved in phenylpropene and monoterpene metabolism in basil (Ocimum basilicum). In this case, EST libraries were generated from several varieties of basil with distinct trichome metabolic profiles, either with high phenylpropene and low terpene content, or the reverse (Gang et al., 2001, 2002b; Iijima et al., 2004a,b). This allowed the successful identification of several key enzymes involved in monoterpene, sesquiterpene and phenylpropene metabolism in these basil varieties (Gang et al., 2002a,b; Iijima et al., 2004a; Koeduka et al., 2006; Vassao et al., 2006; Kapteyn et al., 2007). In total, over 23 000 ESTs were assembled resulting in 7963 unigenes. A complete analysis of these EST databases together with a large-scale proteomics study (881 unique proteins identified) allowed the comparison of the transcriptome and the proteome of basil peltate trichomes (Xie et al., 2008). Several interesting observations could be drawn out of this analysis. In general, there is a good correlation between protein levels and transcript levels, with the most abundant proteins corresponding to the most abundant ESTs. However, 118 of the proteins could not be assigned to any EST, suggesting that the EST dataset of almost 8000 unigenes falls short of covering the proteome that is represented by the most abundant proteins. The availability of four different basil lines with distinct metabolic profiles allowed assessment of the expression of distinct pathways (phenylpropanoid versus terpenoid) and their regulation in a similar or almost identical genetic background. For example, line EMX-1, a poor terpenoid producer, shows drastically reduced transcript and protein levels for the first two enzymes of the MEP pathway, deoxy-xylulose phosphate synthase (DXS) and deoxy-xylulose phosphate reductase (DXR). Interestingly, other steps of the pathway (e.g. the hydroxy-methylbutenyl diphosphate synthase or GCPE) do not seem to be down-regulated, indicating that this differentiation may have occurred fairly recently, only affecting key steps of the pathway. Additionally, the combined transcriptomics–proteomics analysis indicated that the mevalonate pathway is expressed at extremely low levels compared to the plastidic MEP pathway, implying that the cytosolic sesquiterpene biosynthesis relies on IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) pools originating almost exclusively from the plastidic MEP pathway. This is in agreement with previous observations that the MEP pathway contributes to specialized sesquiterpene biosynthesis in some species, such as snapdragon (Antirrhinum majus) and Arabidopsis thaliana (Kasahara et al., 2002; Dudareva et al., 2005).

Table 1.   A list of available glandular trichome-specific expressed sequence tag (EST) collections from plants
SpeciesAccessionTrichome typeSequencingNo. of ESTsReferences
Solanum habrochaitesLA1777MixedSanger2 6561, 2
S. habrochaitesPI126449MixedSanger5 4943
Solanum lycopersicumNAMixedSanger7 2544
Solanum pennellliiLA716MixedSanger2 9172
S. lycopersicumLA3475Mixed stemsNGS278 0005
S. lycopersicumLA3475Type VIISanger7915
S. lycopersicumLA3475Type VINGS225 0005
S. lycopersicumLA3475Type ISanger8315
S. habrochaitesLA1777Mixed leavesNGS108 0005
S. habrochaitesLA1777Type ISanger9785
S. habrochaitesLA1777Type IVSanger1 4255
S. habrochaitesLA1777Type VINGS224 0005
S. habrochaitesPI126449Type VISanger15 0005
Solanum pimpinellifoliumLA1589Type VINGS227 0005
S. pennellliiLA0716Type IVSanger1 2775
S. pennellliiLA0716Type VISanger1 1375
S. pennellliiLA0716Mixed leavesNGS275 0005
S. arcanumLA1708Mixed stemsNGS415 0005
S. lycopersicumMoneymakerMixedNGS195 3776
S. habrochaitesPI127826MixedNGS182 3866
Mentha × piperita PeltateSanger1 3167
Ocimum basilicum PeltateSanger1 3448
O. basilicum PeltateSanger4 8049
O. basilicum PeltateSanger5 42210
O. basilicum PeltateSanger7 31411
Artemisia annua Glandular trichomesSanger24 94712
A. annua Glandular trichomesNGS406 04413
Cannabis sativa Glandular trichomes from female inflorescencesSanger1 54014
Cistus creticus Glandular trichomesSanger2 00415
A. annua Glandular trichomes subtractive librarySanger9 32216
Humulus lupulus Glandular trichomeSanger10 59917
H. lupulus Glandular trichome from female flowerSanger12 36018
Medicago sativa Stem glandular trichomesSanger965919
Medicago truncatula Glandular trichomesSanger10 37720
Nicotiana benthamiana Glandular trichomesSanger6 68621
Nicotiana tabacumBurley 21Glandular trichomesSanger1 94922
N. tabacumK326Glandular trichomesSanger1 16122
N. tabacumSamsunGlandular trichomesSanger3 17922
N. tabacumK326Glandular trichomesSanger5 13923
N. tabacumXanthiGlandular trichomes with or without CdSanger2 10024
Salvia fruticosa Glandular trichomesSanger1 45925

As can be seen from Table 1, certain species have been the focus of important efforts to uncover the transcriptome of their glandular trichomes. Prominent among these are tomato (S. lycopersicum and related wild species) and A. annua, the plant producing artemisinin. Since artemisinin is produced in leaf glandular trichomes, trichome EST sequencing efforts have allowed the discovery of key biosynthetic enzymes, in particular the oxidizing steps after the committed sesquiterpene synthase step (Covello, 2008).

Over the years a major effort has been devoted to producing large-scale EST sequences from tomato trichomes by different research groups, with an estimated total of over 2 million ESTs, all tomato species included (Table 1) (van Der Hoeven et al., 2000; Fei et al., 2004; Fridman et al., 2005; Slocombe et al., 2008; Besser et al., 2009; Dai et al., 2010; Schilmiller et al., 2010a,b; Bleeker et al., 2011; McDowell et al., 2011). One reason for the interest in tomato glandular trichomes is the role that their secretions play in the resistance to a number of insect pests, including white flies (Bemisia sp.) or Tuta absoluta (Simmons and Gurr, 2005; Bleeker et al., 2009). Wild species such as Solanum habrochaites and Solanum pennellii, show much higher resistance levels than the cultivated tomato, and in many cases this could be traced down to glandular trichome secretions (Simmons and Gurr, 2005). Therefore, these species have been the focus of particular attention with regards to their trichome secretions and several transcriptomics studies have been published on the topic in the last decade (van Der Hoeven et al., 2000; Fridman et al., 2005; Slocombe et al., 2008; Ben-Israel et al., 2009; Besser et al., 2009; McDowell et al., 2011). This has created useful resources for the elucidation of the biosynthetic pathways for compounds specific to the glandular trichomes, such as methyl ketones (Fridman et al., 2005; Ben-Israel et al., 2009; Yu et al., 2010), monoterpenes (Schilmiller et al., 2009) and sesquiterpenes (van Der Hoeven et al., 2000; Sallaud et al., 2009). Up to seven different trichome types were described on the aerial parts of tomato plants (Luckwill, 1943), and from those two particular classes of glandular trichomes seem to play major roles in the production of compounds which mediate interactions with insects. Type 4 trichomes, of the capitate type with one to several glandular cells atop a multicellular stalk, produce glucose esters of short chain fatty acids in S. pennellii (Fobes et al., 1985; Slocombe et al., 2008). Type 6 trichomes, consisting of four glandular cells on one plane, produce either terpenes (monoterpenes and sesquiterpenes) or methylketones (MKs). Recent publications on the characterization of tomato terpene synthases indicate that some of them (SlTPS3, SlTPS9 and SlTPS17) are preferentially expressed in stem trichomes, indicating that trichome metabolic profile may be under organ-specific control (Bleeker et al., 2011; Falara et al., 2011).

Metabolic Pathway Discovery

  1. Top of page
  2. Summary
  3. Introduction
  4. Techniques for Collecting and Purifying Trichomes
  5. Glandular Trichomes: Secreting and Producing Glands
  6. Transcriptomics and Proteomics of Glandular Trichomes
  7. Metabolic Pathway Discovery
  8. Metabolic Profiling of Glandular Trichomes
  9. Glandular Trichome Modeling
  10. Future Directions
  11. Acknowledgements
  12. References

The specificity and the high-level expression of the trichome pathways combined with the availability of significant EST resources (see above) has greatly enhanced the discovery and characterization of genes involved in those pathways. A pioneering example is that of the menthol pathway, which, except for one step, was completely elucidated. Since this pathway has been extensively described in excellent reviews, the reader is referred to the most recent of them (Croteau et al., 2005). Identification of the early steps of the artemisinin pathway has also greatly benefited from trichome EST databases, and again this topic is covered in great detail in recent articles, to which the reader is referred (Covello et al., 2007; Covello, 2008; Teoh et al., 2009; Zhang et al., 2009b; Brown, 2010). I will focus here instead on trichome pathways which have been little reviewed but present a particular interest because of their originality. The first concerns the tomato MK pathway which was mentioned earlier. Methyl ketones are fatty-acid-derived molecules and two enzymes could be found to play decisive functions in the last steps from the 3-ketoacyl-acyl carrier proteins (ACPs). ShMKS2, encoding a protein with similarity to bacterial thioesterases, was shown to catalyze the hydrolysis of the 3-ketoacyl-ACP thioester bond, while ShMKS1, a protein of the α/β-hydrolase superfamily, could catalyze the decarboxylation of the resulting 3-keto fatty acids (Ben-Israel et al., 2009; Yu et al., 2010). ShMKS1 shares similarity to tomato methyl jasmonate esterase (LeMJE) as well as methyl salicylate esterase from tobacco. These intriguing connections suggest a recent diversification of this enzyme family in the biosynthesis of defense-related compounds (Fridman et al., 2005). The second pathway concerns the class II sesquiterpenes α-santalene and β-bergamotene from the wild tomato Solanum habrochaites. The sesquiterpene synthase, ShSBS, is a member of the family of plant kaurene synthases, which were, up until then, known to be involved in the biosynthesis of gibberellins and related polycyclic labdanoid diterpenes (Sallaud et al., 2009). More intriguingly, the substrate of ShSBS is not the usual trans, trans-farnesyl diphosphate (E,E-FPP), but rather its cis isomer Z,Z-FPP, itself produced by a short-chain cis-isoprenyl diphosphate synthase called zFPS. Both proteins are targeted to the chloroplast, strongly suggesting that the MEP pathway supplies the isoprenyl precursors for these sesquiterpenes (Sallaud et al., 2009). Similarly, it was found that in cultivated tomato (S. lycopersicum) the major monoterpene, β-phellandrene, is synthesized by related enzymes. The monoterpene synthase, SlPHS1, is a kaurene synthase-like terpene synthase, which shares high levels of sequence identity with ShSBS (Schilmiller et al., 2009). Its substrate, neryl diphosphate, the cis isomer of GPP, is synthesized by a short-chain cis-isoprenyl diphosphate synthase highly similar to zFPS. More recently, this sub-family of kaurene synthase-like enzymes was expanded by the identification of the tobacco cis-abienol synthase, which is also expressed specifically in glandular trichomes and involved in the biosynthesis of the labdanoid cis-abienol (Sallaud, C. and Tissier, A. et al., unpublished data). This high sequence similarity between this tobacco diterpene synthase, using 8-hydroxy-copalyl diphosphate as a substrate, and the tomato ShSBS and SlPHS1, which use acyclic cis-isoprenyl diphosphates, provides interesting material to study terpene synthase substrate specificity, an area that is still poorly investigated. The elucidation of both pathways (MKs and class II sesquiterpenes) was largely facilitated by their trichome specificity and the high-level expression of the corresponding genes and provide good examples of the value of glandular trichomes for gaining insight into novel enzymatic activities.

Metabolic Profiling of Glandular Trichomes

  1. Top of page
  2. Summary
  3. Introduction
  4. Techniques for Collecting and Purifying Trichomes
  5. Glandular Trichomes: Secreting and Producing Glands
  6. Transcriptomics and Proteomics of Glandular Trichomes
  7. Metabolic Pathway Discovery
  8. Metabolic Profiling of Glandular Trichomes
  9. Glandular Trichome Modeling
  10. Future Directions
  11. Acknowledgements
  12. References

The location of trichomes on the surface offers the possibility of recovering trichome-borne secretions by simple solvent washes without extracting internal leaf metabolites. This is a highly efficient and simple method, which restricts the complexity of the extract to compounds located on the surface of the plants provided extraction times remain short, i.e. one to a few minutes. Since many compounds synthesized by trichomes are hydrophobic, organic solvents such as hexane, heptane, methylene chloride, methanol, ethanol or acetonitrile are often used. Depending on the chemical characteristics of the compounds, different analytical methods may be used. A robust method, but with relatively poor resolution, is thin layer chromatography (TLC). Identification of the compounds can only be done by comparison with purified authentic standards, and even so assignments have to be done with care. The advantage of TLC is that it does not require extensive sample preparation and crude extracts can be loaded without fear of damaging the equipment. High-performance TLC (HP-TLC) instruments allow a much higher reproducibility and sensitivity and can even be coupled to a mass spectrometer; alternatively plates may be analyzed by matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) for the identification of compounds (Fuchs et al., 2008). The large choice of solid phases combined with appropriate mobile phases offers a virtually endless continuum of separation conditions. In early reports of tobacco trichome exudates, TLC was often used in combination with gas chromatography (GC) to rapidly evaluate sample composition (Keene and Wagner, 1985; Kandra and Wagner, 1988; Kandra et al., 1990).

However, since in many cases glandular trichomes produce compounds that are volatile or semi-volatile in ambient conditions, GC is often the method of choice for analyzing them. It should be noted that GC also offers flexibility in terms of sample preparation, injection, solid phase and detection. Detection is typically done either with flame ionization detection (FID) or electron impact (EI) ionization followed by mass spectrometry, in general with a simple quadrupole instrument, although GC-TOF and GC-triple quadrupole MS coupling are also available. With FID, compound identification will be essentially done by comparison of the retention time with an authentic standard, or by comparison to the Kovats retention index. This is fairly accurate but not error-proof, particularly in the case of enantiomers. These may, however, be resolved by the use of chiral columns. Gas chromatography-MS offers an extra level of confidence for compound identification by comparison of EI mass spectra with those from a database (e.g. National Institute of Standards and Technology, NIST), but relying solely on MS comparison is certainly not advised to ascertain compound assignment.

The wide range of suitable injection techniques in GC may have a considerable impact on compound detection and identification. The most widely used injection technique is the splitless, whereby samples are first vaporized in the injector, then recondense at the top of the column. Although convenient and fairly trouble-free, this method is not adapted for compounds which are temperature-sensitive and will undergo rearrangements due to the increased temperature during the injection. A typical example of these are the germacrene sesquiterpenes, which can be thermally converted to elemenes (van Der Hoeven et al., 2000; Schilmiller et al., 2010b). To avoid this phenomenon, cool on-column injection (OCI) should be used. In this injection technique, the sample is directly injected onto the column and the temperature is increased progressively to proceed with the separation. Although thermal degradation can still occur during the separation, this method results in much less degradation and produces chromatograms that better reflect the composition of the extract.

To gain a more comprehensive overview of trichome metabolism, in particular primary metabolism, with GC-MS, sample derivatization would be required. However, to the best of my knowledge, this has not been done on any of the major plants bearing glandular trichomes. One issue is the difficulty in collecting a sufficient amount of trichome-specific material for an exhaustive analysis. An alternative to GC is LC-MS metabolic profiling, which offers the advantage of increased sensitivity. This was used as a non-targeted analytical method (LC-TOF) in an investigation of tomato glandular trichomes (McDowell et al., 2011). In this study, exudates from specific trichome types (1, 4, 6 and 7) and from different tomato species (S. lycopersicum, S. habrochaites, S. arcanum, S. pennelllii, S. pimpinellifolium) were analyzed and the results processed by a clustering analysis. Intriguingly, there were fewer differences between trichome types within a species than there were amongst the same types between species. However, this result should be taken with caution since the authors themselves admit that ‘pure’ trichome preparations could not be guaranteed and that cross-contamination between exudates from different trichome types could not be excluded. Liquid chromatography MS was also used as a targeted analytical method for the measurement of flavonoids in trichomes of S. habrochaites (accession LA1777). Methylated myricetins were detected preferentially in the type 1 and 4 trichomes, rather than in Type 6 trichomes. This is consistent with the observation that Type 1 and Type 4 trichomes look much alike, the difference between them being their size (Schmidt et al., 2011). However, it should be noted that Type 6 trichomes also produced significant amounts of those myricetin derivatives. This confirms the data of McDowell et al. (2011) and suggests that, although the different trichome types have distinct profiles, there is also a significant metabolite overlap between them, indicating the presence of common regulatory mechanisms. In both reports (McDowell et al., 2011; Schmidt et al., 2011), exudates were collected from individual trichomes with glass capillaries. The non-targeted analysis described in McDowell et al. (2011) did not show any primary metabolites. Since primary metabolites should be present in lower quantities than the specialized compounds secreted by the trichomes, this may be because insufficient amounts of metabolites were analyzed or because the MS method did not allow for a comprehensive coverage of such metabolites. This is a relevant issue if one wishes to understand how these cell factories achieve such high metabolic fluxes towards restricted classes of compounds, in particular how these specialized metabolic pathways are connected to primary metabolism.

Glandular Trichome Modeling

  1. Top of page
  2. Summary
  3. Introduction
  4. Techniques for Collecting and Purifying Trichomes
  5. Glandular Trichomes: Secreting and Producing Glands
  6. Transcriptomics and Proteomics of Glandular Trichomes
  7. Metabolic Pathway Discovery
  8. Metabolic Profiling of Glandular Trichomes
  9. Glandular Trichome Modeling
  10. Future Directions
  11. Acknowledgements
  12. References

Modeling can be applied to simulate developmental processes or metabolic fluxes. Discrepancies between model predictions and real-life data may be used to iteratively optimize the models and improve their predictive ability. Trichome patterning has been explored in depth in A. thaliana, and a model based on interacting activator and inhibitor complexes has been proposed (Digiuni et al., 2008; Pesch and Hulskamp, 2009; Balkunde et al., 2010). To the best of my knowledge, no patterning models for glandular trichomes have been reported so far. This is probably due on one hand to the lack of genetic and molecular data concerning glandular trichome development and on the other hand to the complexity of patterns, with often several types of glandular trichome (see Figure 1). What has been observed is the apparent competition between stomata and trichomes, or between trichome types, suggesting that common regulatory mechanisms may underlie the patterning of these epidermal differentiations (reviewed in Martin and Glover, 2007). There is a single reported attempt at metabolic modeling of glandular trichomes of mint (Rios-Estepa et al., 2008, 2010). Peppermint glandular trichomes make an interesting model in this respect, because the pathway to the major monoterpenes is well known, enzyme kinetic properties for the corresponding steps are available and in addition the pathway has branches, which bring an additional layer of complexity (Croteau et al., 2005). Moreover, the monoterpene profile changes during the course of plant development, implicating a dynamic regulation of the pathway (McConkey et al., 2000). The first model included a number of parameters such as trichome number, volume of the trichome cells and their subcuticular storage space, amount of monoterpenes present in each trichome, enzyme activity levels and kinetic values (Rios-Estepa et al., 2008). Using those parameters, a set of differential equations was established to simulate monoterpene production in peppermint peltate trichomes. The first simulations were then iteratively optimized by comparing the simulated values with experimental results, leading to a basic model for unstressed conditions. Then, to validate the robustness of the model, stress conditions which alter the secondary compound profile of the essential oil were simulated and tested experimentally. Reduced light led to a 50% reduction of total oil yield and to a modification of the dynamic profile with a transient increase in pulegone and a hyperbolic accumulation of menthofuran, which is a side product of the pathway branching out from pulegone. Previous studies using transgenic plants overexpressing menthofuran synthase (MFS) had pointed to a possible role of menthofuran in the inhibition of pulegone reductase (PR) expression, the committed step to menthol from pulegone (Mahmoud and Croteau, 2001). When simply modulating the expression levels of MFS and PR in the model, no satisfactory simulation could be obtained. However, when inhibitory effects of menthofuran on PR enzyme activity were included, a much better fit could be reached with a competitive inhibition mechanism. This was then further tested experimentally by demonstrating that menthofuran is indeed a competitive inhibitor of PR (Rios-Estepa et al., 2008). Further, it was shown that menthofuran accumulated in the glandular cells and was poorly transported to the storage space, lending additional support to its function as an intracellular inhibitor of PR (Rios-Estepa et al., 2008). In a subsequent study, these authors updated their model by taking into account further experimental data obtained from wild-type plants grown under various environmental stresses (reduced fertilizer, reduced light and reduced light with higher night temperature) and from transgenic plants with reduced levels of MFS (Mahmoud and Croteau, 2001) grown in the same conditions. The model was expanded by taking into account enzymes of the isoprenoid precursor pathway, dynamics of glandular trichome size and density, estimating enzyme concentrations from real-time PCR data, and feedback control of two enzymes (GPS and PR) by products of the pathway (Rios-Estepa et al., 2010). This expanded model was then used to predict essential oil composition and yield from two transgenic lines, one with reduced MFS expression and the other with reduced limonene-3-hydroxylase expression. Two main conclusions could be drawn from this study. First, variations in gene expression levels have an impact on essential oil composition, but relatively little on total oil yield. Second, a determining factor in oil yield is trichome density and size. The fits of the model were in good agreement with the experimental data with non-significant differences as validated with chi-square tests. However, it is difficult to estimate the true predictive ability of the model. A better test would be to predict outcomes in conditions or transgenic plants, which were not tested, and compare those a posteriori. For now, one of the limitations of the model may lie in the relatively limited set of enzymes and pathway steps which it includes. Indeed, only the isoprenoid precursor MEP pathway and the menthol pathway were taken into account. It is likely, however, that primary metabolism plays a crucial role, since the precursors of the MEP pathway, glyceraldehyde-3-phosphate and pyruvate, are directly connected to glycolysis. A more comprehensive model should probably include the primary metabolism network and influx of photosynthetates into the glandular trichome. This in turn could be correlated to growing conditions (light, temperature, nutrient availability). However, the required data sets are missing and it would necessitate a combination of transcriptomics and metabolomics, including fluxomics, to allow the elaboration of a more comprehensive model.

Future Directions

  1. Top of page
  2. Summary
  3. Introduction
  4. Techniques for Collecting and Purifying Trichomes
  5. Glandular Trichomes: Secreting and Producing Glands
  6. Transcriptomics and Proteomics of Glandular Trichomes
  7. Metabolic Pathway Discovery
  8. Metabolic Profiling of Glandular Trichomes
  9. Glandular Trichome Modeling
  10. Future Directions
  11. Acknowledgements
  12. References

So far, glandular trichomes have mostly been studied to elucidate the biochemical pathways of the major compounds they produce and secrete. While this has allowed significant contributions to be made in the field of specialized plant metabolism, particularly in the terpenoid, lipid and phenylpropanoid areas, a deeper understanding of the development, regulation and wiring of the metabolic networks in these specialized cell factories is required to fully exploit their potential. A number of research directions are proposed below to fill these gaps and to make glandular trichomes the fully accomplished research topic that they deserve to be.

A model species for glandular trichome studies?

Arabidopsis has become the reference for molecular genetic studies of non-glandular trichome biology. Similarly a model species for glandular trichome research would also contribute to significantly increase our knowledge in this area by bringing together efforts from laboratories over the world. Although Lamiaceae species like mint or basil appear to be potential candidates because of the early work on glandular trichomes, they suffer from a number of difficulties, especially in the case of mint. These include poor genetics due to the polyploid nature of many cultivated mint species and lack of genomic sequences. The reduced costs and increased throughput of the latest sequencing technologies could, however, overcome this barrier. Recently, Mentha longifolia, a diploid mint ancestor of Mentha spicata has been proposed as a model for mint genetic research (Vining et al., 2005). Similar problems (lack of genetic resources, difficult transformation) concern other plants such as the Cannabaceae (cannabis, hops). Artemisia annua also has the potential to become a model for glandular trichome biology. A genetic map and a mutant collection were developed in the frame of the Center for Novel Agricultural Products (UK) Artemisia project (http://www.york.ac.uk/org/cnap/artemisiaproject/; Graham et al., 2010). In addition, genetic transformation, although not routine, is feasible as attested by several recent publications (Han et al., 2006; Wang et al., 2007; Yang et al., 2008; Feng et al., 2009; Zhang et al., 2009a; Banyai et al., 2010; Alam and Abdin, 2011; Chen et al., 2011; Liu et al., 2011; Nafis et al., 2011). Further, A. annua is related to sunflower (Helianthus annuus), an important crop plant, for which important genetic resources are available. However, the genetic resources of Artemisia itself are still relatively limited and the Artemisia project mentioned above is an effort from a single institute which, although impressive, would benefit from the contribution of the international community, for example by undertaking the sequencing of the Artemisia genome.

In contrast to the plants mentioned above, tomato, as a horticultural crop of worldwide importance, has benefited from many years of research effort in breeding, physiology, development and molecular biology. Therefore, tomato currently looks like a strong candidate to serve as a reference for the field of glandular trichome biology. Its genome sequence is now available, genetic resources are almost second to none with mutant collections, introgression lines derived from crosses with related wild species, and already rich EST databases of trichome-specific sequences are available (see above). In addition, tomato is easily transformed, further expanding the list of tools for probing and validating gene functions in vivo. The species suffers from a lack of exhaustive insertion mutant collections (with T-DNA or transposons), although it should be pointed out that a significant, albeit partial, insertion collection based on Ac/Ds insertions is already developed (Meissner et al., 2000). Yet, a large-scale insertion mutant collection is an effort which would benefit tomato scientists as a whole. TILLING-based or equivalent resources are also available for tomato and already provide adequate reverse genetics populations (Gady et al., 2009; Piron et al., 2010; Sreelakshmi et al., 2010). Moreover, virus-induced gene silencing (VIGS) works well in various tomato species, including for genes which are specifically expressed in trichomes, and may thus be used to rapidly probe gene function (Besser et al., 2009; Becker and Lange, 2010). Additionally, the availability of a ‘miniature’ tomato line, the Micro-tom variety, also makes it possible to perform genetic screens with limited greenhouse space (Meissner et al., 1997; Dan et al., 2006) and several mutant resources have already been generated in this line (Meissner et al., 2000; Okabe et al., 2011; Saito et al., 2011). Thus, exhaustive genetic screens for glandular trichome-related phenotypes are definitely within reach. Studies in tomato could also benefit from work in other Solanaceae, in particular tobacco (N. tabacum). Many pioneering glandular trichome studies were done in this species, for which extensive EST resources and a partial genomic sequence are available. Moreover, it should be pointed out that the genome of 100 Solanaceae species will be sequenced (http://solgenomics.net/organism/sol100/view) including S. pennellii, Nicotiana sylvestris, N. tabacum, N. benthamiana and many others. Through this unique resource, cross-species comparisons will be made easier, allowing the identification of genes which determine distinct glandular trichome types or secretions. Thus, rather than tomato alone, the Solanaceae as a whole – thanks to its diversity, growing genetic and sequence resources – may emerge as the reference plant family to uncover basic processes of glandular trichome biology. However, given the diversity of glandular trichomes and their secretions, it can be argued that no single species will serve as a single model. Rather, certain species or groups of species will emerge as references for certain types of trichomes and corresponding secretions, such as Lamiaceae for capitate trichomes and their capacity to store volatiles, or Solanaceae for capitate trichomes. A summary of the main contending species with their main pros and cons is provided in Table 2.

Table 2.   A summary of characteristics, advantages and difficulties of the major plant species which could serve as models for the study of glandular trichomes
Common nameFamilyLatin names of main and related speciesType of trichomesMain applied interestAdvantagesDifficulties
  1. EST, expressed sequence tag.

MintLamiaceaeMentha × piperita; M. spicataPeltate trichomes which accumulate volatiles Peltate trichomes which accumulate volatilesEssential oil industry. Concerns other related species (Sage, Lavender, Rosemary, Thyme, Oregano)Good background knowledgeDearth of genetic resources. Polyploid species. Propagation and stability through seeds difficult. No genome sequences.
BasilLamiaceaeOcimum basilicumDearth of genetic resources. No genome sequences
TomatoSolanaceaeSolanum lycopersicum; S. pennellii; S. habrochaitesCapitate and Type 6 (similar to peltate trichomes of Lamiaceae)Pest resistanceLarge EST resources; sequenced genome; extensive genetic (forward and reverse) resources; genetic transformation; large scientific community 
TobaccoSolanaceaeNicotiana tabacum; N. sylvestris; N. glutinosaCapitate trichomesPest resistance; precursors of fragrance compounds (Labdane diterpenoids)Large EST resources; partially sequenced genome; genetic transformationRelative lack of easily accessible forward and reverse genetic resources
Sweet wormwoodAsteraceaeArtemisia annuaBiseriatePharmaceutical ingredientsLarge EST resources; genetic map; mutant collection; related to sunflowerSmall community (but growing); limited genetic resources; no genome sequences
MarijuanaCannabaceaeCannabis sativaMulticellular glands with sub-cuticular storage spacePharmaceutical ingredientsEST resourceDioecious species; no genome sequence; genetic transformation not routine; ‘Drug’ status
HopsCannabaceaeHumulus lupulusMulticellular glands with sub-cuticular storage spaceAroma ingredientsEST resourceDioecious species; no genome sequence; genetic transformation not routine
AlfalfaFabaceaeMedicago sativaCapitate trichomesPest resistanceEST resourceTetraploid species; genetic transformation difficult
Barrel MedicFabaceaeMedicago truncatulaCapitate trichomes (few)NDEST resource; genetics; genome sequenceFew glandular trichomes; genetic transformation difficult

Development

Investigations of non-glandular trichomes in the model species Arabidopsis have been very fruitful and have provided a molecular genetic framework for the development of specialized organs (Balkunde et al., 2010). Equivalent approaches for glandular trichomes are missing, in part because of the absence of model plant species (see above). There is some indication that the development of glandular and non-glandular trichomes may be under the control of distinct transcription factor networks (Serna and Martin, 2006). This suggests that simply looking for homologs of Arabidopsis genes involved in trichome development in species with glandular trichomes may not prove a successful strategy, and therefore dedicated studies in the species of interest are required to tackle this issue. Understanding how glandular trichomes develop and how their distribution on the surface of the aerial parts of the plants is controlled will open up new possibilities for engineering and breeding glandular trichome productivity and density. Specific types of glandular trichomes seem to have a rigidly defined developmental plan with a constant number of cells and cell types. For example, the tomato Type 6 trichomes always have four glandular cells, one intermediate cell, a stalk cell and a basal cell (Figure 3). A similar organization is seen in Lamiaceae peltate trichomes, although the intermediate cell is absent and the stalk cell is much shorter (Turner et al., 2000). If glandular trichomes are to be considered as plausible biofactories for the production of renewable chemicals (Schilmiller et al., 2008), significant efforts will have to be devoted to increase their productivity, for example by increasing trichome density or size. Glandular trichome density appears to be under the control of numerous environmental and hormonal factors. In particular, jasmonates seem to play an important role, as confirmed by several independent reports (Thaler et al., 2002; Ament et al., 2004; Li et al., 2004; Boughton et al., 2005; Maluf et al., 2007). However, the signaling pathways are still largely unknown. In addition to genetic screens, which can be performed, for example, on tomato as mentioned above, novel EST resources should be devoted to specific stages of development. Because most of the interest has focused on elucidating biosynthetic pathways, glandular trichome EST libraries have been generated up to now from mature and actively secreting glands. Gathering EST sequences from early and distinct phases of development of glandular trichomes would be one step towards this goal. Since early phases of trichome development are intimately linked to leaf development, the current techniques for trichome isolation will most likely prove irrelevant. This will require establishing novel methods to isolate homogeneous populations of developing trichomes at specific phases of development. This could be done for example by laser microdissection of developing glandular cells or, once a promoter for early phases of trichome development is available, by flow cytometry of leaf protoplasts from a plant expressing a fluorescent protein under the control of this promoter. Such methods have proved successful in characterizing transcriptomes of specific plant cell types (Chen et al., 2010a; Matas et al., 2010; Sozzani et al., 2010; Schiebold et al., 2011). Access to transcriptomes of specific development phases of glandular trichomes will allow us to probe the function of putative transcription or other regulatory factors by reverse genetics strategies.

image

Figure 3.  Initiation and developmental stages of tomato Type 6 glandular trichomes from Solanum lycopersicum. Drawing freely adapted from sections visualized by light microscopy. Left: appearance of a trichome initial (TI) in the epidermal layer. Middle: Intermediate developmental stage with the established overall plan of the trichome. BC, basal cell; SC, stalk cell; IC, intermediate cell; pGC, pre-glandular cell. Right: mature trichome. The pre-glandular cell has divided twice to give four glandular cells of equal size.

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Understanding the glandular trichome cell factories

Another key and unique aspect of glandular trichome biology is their ability to synthesize and secrete large amounts of a limited number of metabolites relative to their size (Schilmiller et al., 2008). As such, they qualify as true cell factories, making them relevant targets for metabolic engineering. This suggests that dedicated metabolic networks have been put in place in these cells. To date, however, there is almost no information on how this high level of specialized metabolite productivity is achieved, in particular how primary metabolism contributes to this high metabolic flux. Although existing EST data can be mined to look for cDNAs encoding enzymes of primary metabolism, comprehensive metabolic profiling of trichome content should be performed to address this issue. For example, it could help unravel novel metabolic shunts that are used in those organs to escape the tight regulation that usually controls specialized metabolism in other non-secretory tissues. Better yet would be fluxomics studies whereby the dynamics of metabolite fluxes through primary metabolite pathways could be analyzed. This would require labeling of plants with stable isotopes, for example 13CO2- or 13C-labeled precursors. Sensitive LC-MS measurements, preferably with high mass precision (i.e. TOF instruments) should allow the relative quantification of 12C/13C ratios of known metabolites and contribute to the identification of unknown metabolites. Such methods are already available and have been implemented in plants (Feldberg et al., 2009; Giavalisco et al., 2011). In the dynamic phase, 13CO2 labeling experiments will permit the identification of the photosynthetates which are incorporated into trichome secretions. Such labeling studies have been performed recently on the cyanobacterium Synechocystis sp. allowing the identification of metabolic pathway fluxes in a photosynthetic organism (Young et al., 2011). Subsequently, feeding with specific precursors which have been identified as important flux nodes could be used to further validate the findings of the 13CO2 labeling. The role of the different intracellular compartments will be more difficult to address because of current technical limitations and challenges in combining metabolite quenching and pure organelle isolation. This was recently applied to analyze the vacuolar metabolome of Chara australis, which, however, benefits from an extraordinarily large cell size (Oikawa et al., 2011). The establishment of a flux map of glandular trichomes combined with the analysis of deep RNA sequencing and proteomics data should allow the identification of potential metabolic bottlenecks and of genes which contribute to their high metabolic productivity. Further, the subcellular targeting of pathway enzymes can be determined by expressing fluorescent protein (FP) fusions in transgenic plants, to identify or confirm the compartmentalization of distinct pathway steps. Such FP fusions may also be used to demonstrate protein–protein interactions in planta via fluorescence lifetime imaging microscopy (FLIM)–fluorescence resonance energy transfer (FRET) measurements (Chen et al., 2010b). More generally, high-throughput two-hybrid screens could allow the production of an interactome map, potentially revealing substrate channeling or regulatory networks.

This can be used to model the functioning of the trichome cell factories and may then be transposed to other plant tissues or other organisms. It will most likely require the co-expression of a relatively large number of genes. Here, the recent spectacular advances in synthetic biology for the cloning of large fragments of DNA (see for example Gibson et al., 2010) make it possible to contemplate the introduction of multiple genes in plants or in microorganisms to ‘reconstitute’ a trichome cell factory. In plants, multigene constructs, either with artificial mini-chromosomes or with large T-DNA, are now possible. Up to 150–190 kb of DNA may thus be transferred to plant chromosomes (for review see Birchler et al., 2010; Naqvi et al., 2010).

Transport and storage of metabolites in glandular cells also require careful inspection. Concerning transport, LTPs in particular have been suspected to be involved due to their relatively high representation in trichome EST databases. Up until now, their contribution to the secretion of metabolites had not been demonstrated, but a recent article indicates that in N. tabacum, NtLTP1, a trichome-specific LTP, seems to play a critical role in the secretion of tobacco terpenoids (Choi et al., 2012). ABC transporters are an important class of proteins, which have been shown to be involved in the transport of a variety of metabolites across membranes. Of relevance to glandular trichomes is the identification and characterization of NpPDR1, an ABC transporter from Nicotiana plumbaginifolia, which was shown to support transport of sclareol and is expressed in trichomes, although not specifically (Stukkens et al., 2005). Furthermore, a proteomics study on tobacco trichomes led to the identification of several transporters, including a number from the ABC class (Van Cutsem et al., 2011). However, validation of their role in transport of trichome metabolites awaits experimental evidence. Storage of volatile compounds in subcuticular spaces, as in the peltate trichomes of the Lamiaceae for example, requires not only transport but also a highly restricted and polarized digestion of the apical cell wall of the secretory cells to dissociate the cuticle from the cell wall. Currently nothing is known about this process and the actual composition of cell wall lining this storage cavity, although they could have interesting practical implications beyond the field of plant biology. This process is intimately linked to trichome development and its elucidation will require transcriptomics and functional studies of pre-secretory stages.

In conclusion, plant glandular trichomes constitute a highly active area of research, fueled by interests in the elucidation of the biosynthetic pathways of industrially relevant compounds such as essential oils, pharmaceutical ingredients or substances, which may be used in plant defense. As cellular factories, glandular trichomes are also attractive targets for metabolic engineering and breeding, particularly regarding the possibility of use plants as biofactories for the renewable production of organic compounds. To reach this long-term goal, a multidisciplinary approach including genetic, developmental, transgenic, metabolomic and fluxomic analyses will have to be implemented.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Techniques for Collecting and Purifying Trichomes
  5. Glandular Trichomes: Secreting and Producing Glands
  6. Transcriptomics and Proteomics of Glandular Trichomes
  7. Metabolic Pathway Discovery
  8. Metabolic Profiling of Glandular Trichomes
  9. Glandular Trichome Modeling
  10. Future Directions
  11. Acknowledgements
  12. References

I wish to thank Frank Syrowatka from the Multidisciplinary Centre of Materials Science, Halle (Saale), Germany, for the environmental SEM pictures of tomato leaves and Ana Simonovic and Sladjana Todorović for providing SEM pictures of Tanacetum parthenium florets and Salvia officinalis leaves and Angelos Kanellis for providing pictures of Salvia trichomes. Work in my laboratory is funded by the EU under the project TERPMED (grant number KBBE-227448) and by the Leibniz Institute of Plant Biochemistry. I would also like to thank Bettina Hause, Thomas Vogt and Michael Walter for critical reading, and both reviewers for suggestions which significantly improved the manuscript. I would like to apologize to authors whose work could not be cited due to space limitations. The author declares no competing financial interests.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Techniques for Collecting and Purifying Trichomes
  5. Glandular Trichomes: Secreting and Producing Glands
  6. Transcriptomics and Proteomics of Glandular Trichomes
  7. Metabolic Pathway Discovery
  8. Metabolic Profiling of Glandular Trichomes
  9. Glandular Trichome Modeling
  10. Future Directions
  11. Acknowledgements
  12. References
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