The difference in external appearance of type VI glandular trichomes in S. habrochaites and S. lycopersicum is reflected by a distinct internal architecture
There are a number of reports that document a higher metabolic productivity of glandular trichomes in the wild tomato species S. habrochaites compared to its cultivated relative S. lycopersicum [18, 23]. Two factors can contribute to this difference: a higher density of trichomes and a higher metabolic activity per trichome. We estimated the number of type VI glandular trichomes per leaflet (n = 5) on the adaxial side at 2573 ± 161 cm−2 in S. habrochaites versus 611 ± 171 cm−2 in S. lycopersicum as measured on leaflets that have an area of 1.6 ± 0.2 cm2 and 2.1 ± 0.9 cm2 respectively. However, this alone cannot account for the large difference in the content of metabolites produced by the trichomes which in absolute quantities can exceed 100 fold. Indeed, the amount sesquiterpene carboxylic acids produced by S. habrochaites LA1777 can reach up to 12 mg g−1 FW [19], whereas foliar concentrations of rutin, the most abundant secondary metabolite produced by glandular trichomes in S. lycopersicum, range from 70 to 170 μg.g−1 FW [24]. It was already observed that type VI trichomes in S. habrochaites and S. lycopersicum have a different appearance [25]. In S. habrochaites the glandular head looks round while in S. lycopersicum the contour of four glandular cells can be clearly distinguished. We confirm this difference in shape based on observations made with an environmental scanning electron microscope (ESEM) (Fig. 1). The type VI trichomes of both species have an identical overall architecture with a glandular head, an intermediate cell and a single stalk cell connecting the trichome to the leaf. The trichome sits on top of a single basal cell, whose diameter is slightly larger than that of the stalk cell (Fig. 1). Glandular cells in the round trichomes of S. habrochaites cannot be distinguished from the outside. But the presence of furrows at earlier stages of development (Fig. 1c) indicates that type VI trichomes of S. habrochaites are likely to contain four glandular cells as well. Thin sections of trichomes attached to leaves and direct observations of trichomes on the leaf surface with a fluorescence microscope confirm the presence of four glandular cells in both species (Fig. 2a and b). In addition, these images reveal the presence in S. habrochaites of a large intercellular space where metabolites can accumulate. In contrast, the type VI trichomes of the cultivated tomato have either no or a very small intercellular space, thus leaving little room for the storage of metabolites. ESEM pictures from shoot apex and developing leaves indicate the presence of fully mature trichomes already on leaf primordia (Additional file 1: Figure S1). Trichomes appear first on the abaxial side and subsequently on the adaxial side, where they end up being much denser (Additional file 1: Figure S1). This indicates that trichome initiation and differentiation occur at different times in different locations within a single leaf. Therefore even very young leaves will contain a trichome population of mixed development stages.
To gain further insight into the development and architecture of type VI trichomes, we carried out a number of experiments using fluorescence and electron microscopy, as well as immuno-stainings which will be presented in the following sections.
Fluorescence microscopy of detached type VI trichomes
Trichomes are difficult to observe in close range directly onto the leaf because of the irregular surface of the leaf and the tendency of trichomes to break easily. Thus, we first isolated them using a glass bead beating procedure (see Materials and Methods) inspired from previously published methods [26, 27]. We carried this out both for the cultivated tomato (LA4024) and the wild tomato (LA1777). The isolated trichomes were then directly observed with a fluorescence microscope without further treatment. Distinct development phases can be observed as illustrated in Fig. 3. In both species, the patchy red autofluorescence indicates the presence of chloroplasts throughout the development phases. Over 99 % of the trichomes are at the mature stage, i.e. with four clearly individualized glandular cells (Fig. 3f, l, r and x). This figure however is likely to be an overestimate because the heads of mature trichomes tend to break more easily than those of young trichomes. This is confirmed by the fact that the heads of young trichomes with only one or two pre-secretory cells are always seen with the intermediate cell (Fig. 3g, h, s and t) whereas this cell is not present in detached mature trichomes. In S. lycopersicum, the chloroplasts of trichomes in the mature stage seem equally distributed indicating that the cells occupy most of the volume of the trichome head (Fig. 3r). In contrast, in S. habrochaites, the plastids seem to aggregate at the periphery and are totally absent from the center, delineating the inter-cellular cavity between the glandular cells (Fig. 3f). The difference between the two species is most apparent in the later stages of development, where the constriction between the glandular cells is clearly marked in S. lycopersicum (Fig. 3p-r and v-x) whereas in S. habrochaites the four cells form a rounded shape (Fig. 3d-f and j-l). In the earlier stages, the development follows a very similar path in both species. With this preparation method the earliest stages we could isolate have one cell at the tip surmounting the intermediate cell (Fig. 3a, g, m and s). At that stage the developing trichome head has a diameter of approximately 20 μm. The single cell at the tip undergoes two successive divisions without significant enlargement, leading to a 4-cell head of around 25–30 μm diameter (Fig. 3c, i, o and u). The trichome head then significantly enlarges to reach a size of around 60 μM. The average diameter is 69.5 μm ± 6 (n = 50) in S. habrochaites and 57.0 μm ± 4.9 (n = 50) in S. lycopersicum. In exceptional cases (less than 0.5 %) trichome heads of over 100 μm could be detected in S. habrochaites (Additional file 1: Figure S2). Observations with a fluorescence microscope of live trichomes on the leaf surface reveal that in the most mature stages, the inter-cellular cavity of the trichome head in S. habrochaites occupies around 65 % of the total volume (Fig. 2b), with the remaining cytoplasm forming a thin layer at the periphery of the trichome head. Since in S. habrochaites, the head cells seem to enlarge before the inter-cellular cavity is formed, this indicates that the intracellular volume dramatically decreases as the trichome matures.
Transient accumulation of a flavonoid-derived fluorescent substance in the early stages of trichome development
One striking feature common to both species is the presence of an intense yellow-green auto-fluorescence in the early stages of development, which progressively decreases after the second division to completely disappear in the mature stages (Fig. 3 and Additional file 1: Figure S3). The fluorescence spectrum is reminiscent of a flavonoid-type compound (Additional file 1: Figure S4), although it should be noted that flavonoids typically are poorly auto-fluorescent. Thus it is unlikely that this unknown substance is an unmodified flavonoid. Due to the highly transient presence of the compound and the small numbers of trichomes at early stages of development we have not been able to isolate sufficient quantity of material to identify the compounds yet. Interestingly, in a recent manuscript, tomato mutants in a gene encoding chalcone isomerase (CHI) have smaller type VI trichomes which produce significantly less monoterpenes [28]. Since CHI is a key enzyme in the biosynthesis of flavonoids, it was of particular interest to determine if this fluorescence observed in the WT is still present in this mutant (Solanum lycopersicum accession LA1049). Therefore, trichomes from LA1049 plants were isolated and observed with a fluorescence microscope. Remarkably, the yellow fluorescence is now concentrated in discrete spots (Fig. 4), which are also visible in light microscopy suggesting the fluorescence is concentrated in vesicle-like structures. Observations with a confocal microscope indicate that these vesicles are localized within the cells (Fig. 4f and l). Furthermore, this fluorescent material is visible throughout the development of the trichomes including in trichomes with four head cells, whereas in the wild type, the fluorescence disappears in the mature stages. These observations confirm that the fluorescent compound seen in the wild type is a flavonoid and that it plays a critical role in the correct differentiation of the type VI glandular trichomes into active terpene-secreting structures.
The junction between the head cells and the intermediate cell constitute a fragile point facilitating the release of metabolites
We noted that when type VI trichomes are collected, only the head cells are present, except in the earliest development stages, when the head contains only one or two cells, which remain attached to the intermediate cell. In S. habrochaites, trichomes that are still bound to the leaf have a perfectly round appearance (Fig. 5a). In contrast, detached trichome heads appear as though they are collapsed, with the cell walls between the glandular cells having a wavy appearance (Fig. 5b). These observations point to the role of the intermediate cell as a plug preventing the release of metabolites form the inter-cellular cavity. Furthermore, this also indicates that the junction between the intermediate cell and the head cells constitutes a fragile point favoring the release of the metabolites when the trichomes are physically damaged, for example by an herbivore. Confirmation of this hypothesis was provided by the observation of decapitated trichomes by fluorescence microscopy showing that the breakage takes place between the four head cells and the intermediate cell (Fig. 5c). The intermediate cell exhibits a strong blue fluorescence indicative of the presence of phenolic compounds in the cell wall, and remains attached to the stalk cell. Further evidence of the junction between the head cells and the intermediate cell as a fragile point is provided by electron microscopy images (see below).
Ultrastructure of type VI trichomes
To delve deeper into the structure of the type VI trichomes, ultra-thin sections of apices were observed by transmission electron microscopy. Images of the trichomes from LA4024 and LA1777 still attached to the leaves and at different development stages could be observed and are described below.
The earliest stages of trichome initiation can be traced back to enlarged epidermal cells which bulge out of the surface (Fig. 6a). At this stage, it is not really possible to distinguish between the different types of trichomes that will emerge from these trichome initials. The first signs pointing to the development of type VI trichomes is a first unequal division of the trichome initial, resulting in a small apical cell and the future stalk cell (Fig. 6b). The apical cell divides once again unequally, giving rise to an apical cell and the intermediate cell, although this stage could not be directly observed. As seen in the fluorescence microscopy, the apical cell then undergoes a first round of anticlinal division (Fig. 6c) followed by another giving rise to the four glandular cells. This pattern is identical in S. lycopersicum and in S. habrochaites, indicating a similar developmental program in both species. In the early stages, the apical and the intermediate cells have a dense cytoplasm with one larger vacuole and a few small vacuoles. In contrast the stalk cell has a large vacuole which occupies the majority of the cell volume. All major organelles can be clearly distinguished in the early stages in the apical and intermediate cells, including mitochondria and plastids, where starch granules can also be observed (Fig. 7a, c and Additional file 1: Figure S5). At these stages the nuclei are large and display a central nucleolus in all cells (inserts Fig. 7a, c).
Mature trichomes have common and distinguishing features between the two species. In both species, the number of small vacuoles is increasing and progressively filling up most of the cellular space in the glandular and in the intermediate cells (Fig. 7b and d), although there are more and smaller vacuoles in S. habrochaites than in S. lycopersicum. In the intermediate cell the nucleus is still clearly visible, but the nucleolus is much smaller. Instead, dark nuclear material is now accumulating at the periphery lining the nuclear envelope, which typically corresponds to clumps of condensed chromatin (Fig. 8b and f). These changes seem even more pronounced in the nuclei of the secretory cells of mature trichomes (inserts of Fig. 7b and d). The amount of condensed chromatin in the mature trichomes seems to be much larger than in young trichomes, indicating extensive remodeling of chromosome architecture during trichome differentiation and maturation. Another common feature is the presence of a lump of extracellular material exactly at the junction between the intermediate and the glandular cells (Fig. 8b-c and f-g). This material covers the thick cell wall of the glandular cells and the thinner wall of the intermediate cell. The boundary between the two cell walls is also clearly visible, likely delineating the position where the separation occurs (Fig. 8c and g). Although this material has a different appearance between S. lycopersicum and S. habrochaites, its position and distribution across the boundary between the glandular and the intermediate cells suggest a similar role. The accumulation of such extra cell wall material can be seen in abscission zones [29]. In both species, the chloroplasts in the mature trichomes do not have recognizable thylakoid membranes (Additional file 1: Figure S5B and F). Instead, they contain darker staining patches, particularly in LA4024, which have no apparent organized structure and seem to be squeezed between the dense network of small vacuoles or vesicles. Whether the chloroplasts are degenerating or have an organization which is specific to the trichomes remains to be determined. One major difference between the two species is the size of the inter-cellular space. In S. habrochaites, this space is now occupying a large part of the volume of the glandular head (estimated at 65 %). There is hardly any electron absorbing material, indicating that during preparation of the material for the sections the metabolites that are stored therein have been released or that the metabolites are electron-transparent, but ruling out the presence of polymeric material that would be bound to the cell wall. This cavity is likely to have been formed through hydrolysis of the internal cell wall separating the glandular cells, and remains of this cell wall can still be seen as debris in the earlier stage (Additional file 1: Figure S5C and D) and as a wavy thread going from the top to the bottom of the space in the mature stages (Fig. 2d and Additional file 1: Figure S5D). In contrast, in S. lycopersicum the glandular cells still occupy the majority of the volume, although a small intercellular space is clearly visible. Here, electron absorbing material can be seen, suggesting that cell wall hydrolysis is not as extensive as in S. habrochaites (Fig. 7d). This is also supported by the fact that the internal cell walls lining this space are significantly thicker than in S. habrochaites (Fig. 7d).
Observation of the intermediate cell also reveals common and distinct features between the two species. Plasmodesmata at the proximal side, i.e. between the intermediate and the stalk cells, can be seen in both species (Fig. 8b and f, for details see Additional file 1: Figure S5G). The distal side of the intermediate on the other hand shows differences. In S. habrochaites, the area in contact with the cavity and interfacing the glandular cells appears like an empty space likely to be extra-cellular (Fig. 8b). This space is delimited by a membrane which in a few places comes into contact with the glandular cell. The cell wall lining this area appears loose and stains poorly, indicating a possible degradation. In the corresponding area in S. lycopersicum, a well-structured cell wall is visible, with the presence of plasmodesmata, although not as clearly visible as in the proximal side (Additional file 1: Figure S5H). This could be due to the orientation of the section however. These observations point to common mechanisms for the communication between the intermediate and the stalk cell but to different characteristics concerning the communication between the intermediate and the glandular cells.
Another feature of interest is the outer envelope lining the glandular cells. In both species it has a darker staining thin edge, possibly corresponding to the waxy layer of the cuticle and a more lightly stained thick internal part likely representing cell wall material. In the early stages, it is already quite thick (between 0.6 and 0.8 μM) (Fig. 7a and c) although the cells will significantly enlarge. In mature cells, it has the same thickness, indicating there must have been deposition of material to adjust to the increased surface to cover (Fig. 8d and h).
Immuno-labelling of cell wall components
The presence of an intercellular cavity and of a thick envelope indicates that specific cell wall metabolism and remodeling events are likely to take place during the development of type VI trichomes of S. habrochaites LA1777. To get a first insight into these aspects, several commercially available antibodies recognizing distinct cell wall components were used for immuno-labeling followed by fluorescence microscopy (Fig. 8). The sections were carried out on young leaves in order to observe the trichomes at different stages of development and still attached to the leaves. Methyl-esters of pectin are recognized selectively by JIM7 while it does not bind un-esterified homogalacturonan. JIM7 labelling consistently results in strong labelling of the outer cell wall of the developing and mature trichomes, while the inner cell wall only gives a light signal during the very early stages of development (Fig. 9, top row). In contrast, labelling with LM19, which preferentially recognizes un-esterified homogalacturonan but also binds esterified pectin, gives a strong signal in the inner cell wall, particularly at stages during which formation of the cavity is emerging (Fig. 9, second row from the top and Additional file 1: Figure S6). Thus, pectin demethylation seems to play a role for the lysis of the inner cell wall and formation of the inter-cellular cavity. LM13 specifically recognizes arabinan polymers and gives a striking pattern with a strong labelling of the intermediate cell specifically in the mature stage (Fig. 9, third row from the top and Additional file 1: Figure S6). Finally, LM6 which in addition to arabinans also recognizes arabinogalactan proteins (AGPs) gives a signal on the outer cell wall and a very strong signal in the inner cell wall also at the mature 4-cell stage (Fig. 9, fourth row). This indicates that AGPs are constituents of the outer and particularly the inner cell walls.