Light, conventional and environmental scanning electron microscopy of the trichomes of Cucurbita pepo subsp. pepo var. styriaca and histochemistry of glandular secretory products

BACKGROUND AND AIMS: In the present study, the differences between glandular and non-glandular trichomes, the secretory process and the method of secretion were studied. Previous studies on leaves of Styrian oil pumpkin (Cucurbia pepo var. styriaca) plants have shown that four morphologically and ontogenetically independent glandular and non-glandular trichome types and one bristle hair type can be distinguished. The four types of trichomes can be categorized into three glandular trichome types: type I, a short-stalked trichome with four head cells including a 'middle-cell', two stalk cells and one basal cell; type II, a long-stalked trichome with two head cells, a 'neck-cell' region and a long stalk area; type IV, a 'stipitate-capitate' trichome with a mesophyll cell basement, a short stalk and a multicellular head; type III, a non-glandular 'columnar-digit' trichome, which consists of two head cells continuous with three-celled stalk, and the basal cell. METHODS: The histochemical studies (the main classes of metabolite in secreted material of glandular trichomes) were conducted in fresh and fixed hand sections, using the following tests: Sudan black B, Nile blue A, osmium tetroxide, neutral red, Naturstoffreagent A, FSA (fuchsin-safranin-astra blue), NADI (naphthol + dimethylparaphenylenediamine) and ruthenium red. Each suggested differences in the intercalations during the ontogenetical development of each trichome during the development stage. KEY RESULTS: The histochemical reactions revealed the main components of the materials secreted by all types of trichomes, which include lipids, flavones and terpenes and the different cell wall compositions. Glandular secretions were observed during environmental scanning electron microscopy (ESEM) and the trichomes compared with those seen by conventional scanning electron microscopy (CSEM). CONCLUSIONS: Scanning electron microscopy and histochemical analysis demonstrated that each of the trichomes studied produced and released secretory products in a characteristic way.     

Differences in distribution and performance of two sap-sucking herbivores on glandular and non-glandular Datura wrightii

1. Datura wrightii Regel (Solanaceae) is polymorphic with regard to trichome type. Some plants are densely covered with short, non-glandular trichomes, whereas other plants in the same populations possess glandular trichomes that excrete a sticky exudate. The hypothesis that glandular trichomes enhance resistance to all small insect herbivores is evaluated.
2. Field censuses in four southern Californian D. wrightii plant populations revealed that glandular plants are indeed resistant to whitefly spp. (Homoptera: Aleyrodidae). Whiteflies are almost exclusively found on non-glandular plants. In contrast, Tupiocoris notatus (Distant) (Heteroptera: Miridae), another sap-sucking herbivore of similar body size, is found predominantly on plants with glandular trichomes.
3. Laboratory experiments showed that whiteflies are unable to colonize glandular D. wrightii phenotypes. After the whitefly adults had landed on the leaves of these plants, they were trapped in the exudate and died.
4. Tupiocoris notatus adults, on the other hand, laid significantly more eggs on glandular plants. The presence of the exudate was shown to be the cue that determined their choice of glandular plants.
5. In no-choice experiments, T. notatus nymphs reared on glandular plants had significantly higher survival rates and had shorter developmental periods than those raised on non-glandular plants. This, combined with the higher oviposition rates, resulted in higher T. notatus population growth rates on glandular plants than on non-glandular plants.
6. Glandular trichomes are not therefore a universal protection against small herbivores. Differences in distribution over the two plant types within the natural herbivore guild on D. wrightii may, among other selection pressures, contribute to the maintenance of the observed trichome polymorphism.     

Trichome micromorphology of Iranian Stachys (Lamiaceae) with emphasis on its systematic implication

Trichomes of 37 taxa of the genus Stachys and one species of Sideritis (S. montana) were examined using light and scanning electron microscopy. The indumentum shows considerable variability among different species, but is constant among different populations of one species, and therefore, affords valuable characters in delimitation of sections and species. The characters of taxonomic interest were presence of glandular and non-glandular trichomes, thickness of the cell walls, number of cells (unicellular or multi-cellular), presence of branched (dendroid) trichomes, presence of vermiform trichomes, orientation of trichomes in relation to the epidermal surface, curviness of trichomes, and presence of papillae on trichome surface. Two basic types of trichomes can be distinguished: glandular and non-glandular trichomes. The glandular trichomes can in turn be subdivided into subtypes: stalked, subsessile, or sessile. The stalks of the glandular trichomes can be uni- or multi-cellular. Simple unbranched and branched trichomes constitute two subtypes of non-glandular trichomes. Our data do not provide any support for separation of Sideritis from Stachys. The following evolutionary trends are suggested here for Stachys: vermiform trichomes with stellate base are primitive against vermiform trichomes with tuberculate base, long vermiform trichomes are primitive against the short simple trichomes, appressed trichomes are advanced against spreading ones, and loss of glandular trichomes is advanced against their presence. Overall, trichome micromorphology is more useful in separation of species within sections rather than characterizing large natural groups known as sections, except for few cases.     

A Study on the Morphology of Foliar Trichomes of Evergreen Oaks (Quercus) in China and Its Implication in Taxonomy

Most of the evergreen oaks (Quercus L.) are endemic to China and distributed in a large moutainous area of southwestern China at an altitude of 2600-4000m. The delimitation of sections and species in the group has not been satisfactorily solved. The foliar trichomes are very an important character in identifying species of oaks. As a result of observation on 17 species and 2 varieties of evergreen oaks in China under scanning electron microscope, ten types of foliar trichomes are recognized: Simpleuniseriate, Simple-branched, Bulbous, Rosulate, Solitary, Stipitate-fasciculate, Fasciculate, Multiradiate, Stellate and Fused-stellate. The first four types fall into glandular trichomes and the last six non-glandular trichomes. The taxa examined have a combination of various types of the trichomes. All the evergreen oaks have non-glandular trichomes on their foliar epidermis, but glandular trichomes occur solely in certain taxa. These two types of foliar trichomes are obviously different in structure and function, which represent different adular trichomes and the last six non-glandular en oaks are divided into two groups: the glandular group and the non-glandular group according to the types of foliar trichomes and it is reasonable to divide the evergreen oaks into two sections: Sect. Suber (Reichenb.) Spach and Sect. Englerianae (A. Camus) Hsu et Jen. Foliar trichomes of the evergreen oaks show a continuity in density from species predominately with glandular trichomes at a high altitude to species solely with non-glandular trichomes at a low elevation. The characters of branching of foliar trichomes, especially the number of branches, can imply to some extent the evolutionary position of a given evergreen species. Considering the types, density and branches of foliar trichomes combined the evolutionary relationships among the evergreen oaks in China are proposed. Q. gilliana is the startpoint, from where evolution took place in two directions: glandular and non-glandular. In the glandular direction envolved are Q. aquifolioides, Q. longispica and Q. guyavaefolia. Q. pannosa is a species with the most types and highest number of glandular trichomes. In non-glandular line aligned are Q. spinosa, Q. senescens, Q. senescens var. muliensis, Q. monimotricha with the reduction of glandular trichomes and increase of non-glandular trichomes. After Q. tungmaiensis no glandular trichomes appear and it solely depends on non-glandular trichomes to protect itself. Q. granchetii is the climax in the non-glandular line with the highest density and number of branches of foliar trichomes. And then some species, such as Q. engleriana, Q. phillyraeoides, have few or no trichomes. Q. dolicholepis, Q. baronii and Q. baronii var. capitata show the close re-lationships because they share fused-stellate trichomes.     

6-Benzylaminopurine treatment induces increased pubescence on wheat leaves

The epidermis of wheat (Triticum aestivum L.) leaves contains trichomes that contribute to resistance to insect pests and drought tolerance. In the present study, we examined the effects of 6-benzylaminopurine (BA) and methyl jasmonate (MeJA) treatment on trichome development on the leaves of wheat cv. Norin 61 seedlings. Without phytohormone treatment, trichomes on the adaxial leaf surface were short (90 μm) and their density was low (3.6 trichomes/mm²). Both BA and MeJA treatments significantly increased the density of trichomes, and there were no significant differences between the phytohormone treatments. BA treatment increased trichome length to five times as long as that in the control, whereas MeJA treatment did not significantly affect trichome length. Since BA treatment concurrently increased the DNA content of the nuclei in trichome cells, endoreduplication of the nuclei is probably involved in trichome enlargement. These results indicate that even wheat cultivars with short trichomes retain the mechanisms for trichome enlargement and stimuli such as BA application can induce increased pubescence on wheat leaves.     

Glandular trichomes as a barrier against atmospheric oxidative stress: Relationships with ozone uptake,leaf damage,and emission of LOX products across a diverse set of species

There is a spectacular variability in trichome types and densities and trichome metabolites across species, but the functional implications of this variability in protecting from atmospheric oxidative stresses remain poorly understood. The aim of this study was to evaluate the possible protective role of glandular and non‐glandular trichomes against ozone stress. We investigated the interspecific variation in types and density of trichomes and how these traits were associated with elevated ozone impacts on visible leaf damage, net assimilation rate, stomatal conductance, chlorophyll fluorescence, and emissions of lipoxygenase pathway products in 24 species with widely varying trichome characteristics and taxonomy. Both peltate and capitate glandular trichomes played a critical role in reducing leaf ozone uptake, but no impact of non‐glandular trichomes was observed. Across species, the visible ozone damage varied 10.1‐fold, reduction in net assimilation rate 3.3‐fold, and release of lipoxygenase compounds 14.4‐fold, and species with lower glandular trichome density were more sensitive to ozone stress and more vulnerable to ozone damage compared to species with high glandular trichome density. These results demonstrate that leaf surface glandular trichomes constitute a major factor in reducing ozone toxicity and function as a chemical barrier that neutralizes the ozone before it enters the leaf.     

Exogenous Methyl Jasmonate Alters Trichome Density on Leaf Surfaces of Rhodes Grass (Chloris gayana Kunth)

Jasmonates, including jasmonic acid and its derivatives such as methyl jasmonate (MeJA), are plant growth substances that control various responses. Jasmonates regulate leaf trichome density in dicotyledonous plants, but their effects on the trichome density of monocotyledonous plants, such as those in the Poaceae, remain unclear. In the present study we examined the effects of exogenous MeJA on the trichome density of Rhodes grass, which has three kinds of trichomes: macrohairs, salt glands, and prickles. Exogenous MeJA significantly increased the densities of macrohairs and salt glands on the adaxial and abaxial leaf surfaces and those of prickles on the adaxial leaf surface. Because exogenous MeJA significantly reduced the leaf area, we calculated the number of trichomes per 1000 epidermal cells to eliminate the effects of reduced leaf area. Exogenous MeJA significantly increased the number of macrohairs per 1000 epidermal cells on both adaxial and abaxial leaf surfaces, but it significantly decreased the number of salt glands per 1000 epidermal cells on both surfaces. Exogenous MeJA had no significant effects on the number of prickles per 1000 epidermal cells on either of the leaf surfaces. These results indicate that exogenous MeJA alters the trichome density by affecting leaf area and trichome initiation, and the effects of exogenous MeJA on trichome initiation differ among the various trichome types.     

Localization of salvinorin A and related compounds in glandular trichomes of the psychoactive sage, Salvia divinorum

BACKGROUND AND AIMS: Salvia divinorum produces several closely related neoclerodane diterpenes. The most abundant of these, salvinorin A, is responsible for the psychoactive properties of the plant. To determine where these compounds occur in the plant, various organs, tissues and glandular secretions were chemically analysed. A microscopic survey of the S. divinorum plant was performed to examine the various types of trichomes present and to determine their distribution. METHODS: Chemical analyses were performed using thin layer chromatographic and histochemical techniques. Trichomes were examined using conventional light microscopy and scanning electron microscopy. KEY RESULTS: It was found that neoclerodane diterpenes are secreted as components of a resin that accumulates in peltate glandular trichomes, specifically in the subcuticular space that exists between the trichome head cells and the cuticle that encloses them. Four main types of trichomes were observed: peltate glandular trichomes, short-stalked capitate glandular trichomes, long-stalked capitate glandular trichomes and non-glandular trichomes. Their morphology and distribution is described. Peltate glandular trichomes were only found on the abaxial surfaces of the leaves, stems, rachises, bracts, pedicles and calyces. This was consistent with chemical analyses, which showed the presence of neoclerodane diterpenes in these organs, but not in parts of the plant where peltate glandular trichomes are absent. CONCLUSIONS: Salvinorin A and related compounds are secreted as components of a complex resin that accumulates in the subcuticular space of peltate glandular trichomes.     

Ficus carica L. leaf anatomy: Trichomes and solid inclusions

Ficus carica L., a typical plant of the Mediterranean environment, presents leaves covered by an extensive indumentum, and a mesophyll full of solid inclusions. The morphology and ultrastructure of the trichomes, calcium carbonate cystoliths and silicified structures of leaves of F. carica cv Dottato were investigated with light, confocal, scanning electron microscopy, and energy-dispersive X-ray spectroscopy. At the same time, histochemical reactions were also employed to analyse the indumentum composed by glandular and non-glandular trichomes by applying chemical reagents and fluorescence microscopy. Non-glandular and glandular trichomes, capitate, are described. Non-glandular trichomes are unicellular simple, spine-like and present different morphology and sizes. The capitate glandular trichomes are present on leaf adaxial and abaxial surface and consist of one-celled stalk and 3/4 cells spherical head. Histochemical characterisation of leaf hairs revealed the presence of flavonoids, while glandular trichome head cells showed a complex mixture of alkaloids, essential oil and flavonoids. Cu and Al were found in the constitutive structures, spike and dome, of the cystoliths. Several epidermal cells and non-glandular trichomes were silicified. Leaf hairs, trichomes secretions, solid inclusions and silicification of F. carica leaf have significant roles to play in relation to leaf protection from external factors, including high-intensity radiation, herbivores or pathogens.     

An investigation of the foliar trichomes of Tetradenia riparia (Hochst.) Codd [Lamiaceae]: An important medicinal plant of Southern Africa

The morphology and distribution of leaf trichomes of Tetradenia riparia were studied using light and scanning microscopy. Three morphologically distinct types of trichomes were observed on T. riparia leaf surfaces: glandular capitate (short and long stalked), peltate and non-glandular. The glandular and non-glandular trichomes were present in abundance on both the adaxial and abaxial surfaces. Young leaves were densely covered with trichomes; however, the density of trichomes decreases progressively with leaf maturity. This suggests that the trichomes are established early in leaf differentiation and their density decreases with leaf development and age.     

Comparative analysis of leaf trichome structure and composition of epicuticular flavonoids in Finnish birch species

The morphology, ultrastructure, density and distribution of trichomes on leaves of Betula pendula, B. pubescens ssp. pubescens, B. pubescens ssp. czerepanovii and B. nana were examined by means of light, scanning and transmission electron microscopy. The composition of flavonoids in ethanolic leaf surface extracts was analysed by high pressure liquid chromatography. All taxa examined contained both glandular and non-glandular trichomes (short and/or long hairs) but differed from each other in trichome ultrastructure, density and location on the leaf. Leaves of B. pubescens were more hairy than those of B. pendula, but the latter species had a higher density of glandular trichomes. Of the two subspecies of B. pubescens, leaves of ssp. pubescens had more short hairs on the leaf surface and four times the density of glandular trichomes of leaves of ssp. czerepanovii, whereas, in the latter subspecies, short hairs occurred largely on leaf veins, as in B. nana. The glandular trichomes were peltate glands, consisting of medullar and cortical cells, which differed structurally. Cortical cells possessed numerous small, poorly developed plastids and small vacuoles, whereas medullar cells had several large plastids with well-developed thylakoid systems and fewer vacuoles. In B. pubescens subspecies, vacuoles of the glandular cells contained osmiophilic deposits, which were probably phenolic, whereas in B. pendula, vacuoles of glandular trichomes were characterized by the presence of numerous myelin-like membranes. The composition of epicuticular flavonoids also differed among species. The two subspecies of B. pubescens and B. nana shared the same 12 compounds, but five of these occurred only in trace amounts in B. nana. Leaf surface extracts of B. pendula contained just six flavonoids, three of which occurred only in this species. In summary, the structure, density and distribution of leaf trichomes and the composition of epicuticular flavonoids represent good taxonomic markers for Finnish birch species.     

Molecular markers locate genes for resistance to the Colorado potato beetle,Leptinotarsa decemlineata,in hybrid Solanum tuberosum x S. berthaultii potato progenies

The wild Bolivian potato, Solanum berthaultii Hawkes, has been used as a source of resistance to the Colorado potato beetle (CPB), Leptinotarsa decemlineata Say, one of the most significant pests of potato. In this study, two reciprocal backcross S. tuberosum x S. berthaultii potato progenies, BCB and BCT, were mapped with RFLP markers and screened for resistance to CPB consumption, oviposition and defoliation. The genotypic and phenotypic data were combined and analysed to locate quantitative trait loci (QTLs) for resistance to CPB. Three QTLs on three chromosomes in BCB, and two QTLs on two chromosomes in BCT influenced resistance. The QTLs were generally additive but one instance of epistasis was noted. Each QTL accounted for 4–12% of the phenotypic variation observed in resistance. In the more resistant BCB population, a three QTL model explained ca. 20% of the variation in CPB oviposition. When alleles at the three QTLs were homozygous S. berthaultii, oviposition was reduced ca. 60% compared to the heterozygotes. The QTLs for resistance to CPB were compared to those previously identified for the type A and B glandular trichomes, which have been implicated in resistance in the same progenies. Generally, the QTLs for resistance to CPB coincided with loci associated with the glandular trichomes confirming the importance of the glandular trichomes in mediating resistance. However, a relatively strong and consistent QTL for insect resistance in both BCB and BCT on chromosome 1 was observed that was not associated with any trichome traits, suggesting the trichomes may not account for all of the resistance observed in these progenies.     

The role of glandular and non-glandular trichomes in the negative interactions between strawberry cultivars and spider mite

In tropical countries, spider mite (Tetranychus urticae Koch) is a major pest of strawberries. This pest is mainly controlled by the application of pesticides. Use of pest-resistant cultivars is a healthy and environment-friendly alternative to pesticide use. This paper describes the role of glandular and non-glandular trichomes in the interaction between strawberry cultivars and spider mite. The methodology used in this study was based on two bioassays. First, the thumbtack bioassay of Weston and Snyder was used to differentiate strawberry cultivars in terms of the distance travelled by the mites. Second, different types of trichomes present on the abaxial surface of the strawberry leaves were identified and counted. The results of this study showed a significantly negative correlation between the distance travelled by the spider mites and the density of glandular trichomes on the strawberry plant. The cultivars Camino Real and Dover had the highest densities of glandular trichomes and the shortest distance travelled by the mites. In contrast, the cultivars Ventana and Toyonoka had the lowest density of glandular trichomes and the longest distance travelled by the spider mites. The high density of glandular trichomes might have been responsible for the spider mite non-preference to the Camino Real and Dover strawberry cultivars, whereas the non-glandular trichomes minimally contributed to this non-preference. The results of this study affirm the role of glandular trichomes in negative interactions between strawberry and spider mites.     

Trichome micromorphology of the East Asiatic genus Chelonopsis (Lamiaceae) and its systematic implications

The micromorphology of trichomes of the leaves of 17 taxa (including two varieties) of the genus Chelonopsis Miq. and of six species representing four additional genera (Bostrychanthera deflexa Benth., Colquhounia coccinea Wall. var. coccinea, Co. seguinii Vaniot. var. seguinii, Gomphostemma chinense Oliv. var. chinense, G. crinitum Wall. ex Benth. and Physostegia virginiana (L.) Benth.) was surveyed by light and scanning electron microscopy. Two basic types of trichomes can be identified: non-glandular and glandular trichomes. The non-glandular trichomes can be subdivided into two subtypes: simple unbranched and branched trichomes. Based on the cell number, simple unbranched trichomes are further divided into four shapes (unicellular, two-celled, three-celled, and more than three cells), whilst branched trichomes are separated into three shapes (biramous, stellate, and dendroid trichomes). The glandular trichomes can in turn be subdivided into four subtypes: subsessile, capitate, clavate, and sunken. Non-glandular trichomes with two cells (NGTW) and subsessile glandular trichomes (GSU) are most widespread in all taxa examined. The indumentum shows considerable variation among different sections or species. Consequently, trichome micromorphology and distribution have high taxonomic value for Chelonopsis at both infrageneric and interspecific levels. The presence of capitate glandular trichomes (GCA) provides an additional morphological character to clarify the boundaries between subgenus Chelonopsis and Aequidens Wu and Li. Within subgenus Aequidens, non-glandular trichomes with more than three cells (NGMT) and clavate glandular trichomes (GCL) are important characters for sectional division between sect. Aequidens Wu and Li and sect. Microphyllum Wu and Li. Again, three forms of three-celled trichomes can be used as a distinctive taxonomic character at specific level between C. albiflora Pax et K. Hoffm. ex Limpr., C. forrestii J. Anthony, and C. souliei (Bonati) Merr. in sect. Aequidens. This study supports Wu's delimitation of subgenus and sections and the subsequent review work by Xiang et al. Additionally, distribution of trichome types is correlated with the altitudinal distribution and habitats of some species in Chelonopsis.     

Resource availability and the trichome defenses of tomato plants

We conducted two experiments to determine how resource availability influenced allocation by tomato (Lycopersicon esculentum) to trichomes, and how different patterns of trichome allocation by plants grown in different resource environments might then influence the behavior of tobacco hornworm (Manduca sexta) caterpillars. In the first experiment we used high and low levels of light and water, and then, using scanning electron microscopy, determined trichome densities on the leaves and stems. We sampled leaves and stems at several places throughout the plant to determine whether there were within-plant differences in allocation to trichomes. The results of the first experiment showed that resource availability influenced allocation to trichome growth. Patterns in high and low-light supported both the growth-differentiation balance hypothesis (GDBH) and the carbon-nutrient balance hypothesis (CNBH). However, the GDBH was not supported by differences among water treatments. Contrary, to predictions of the GDBH, plants with intermediate growth did not have the highest trichome densities, and plants with similar growth differed in trichome density. Possible biological and artifactual explanations are discussed. The first experiment also showed that there was within-plant variation in allocation to trichomes, and that plant resource availability may influence within-plant variation in allocation to trichomes. In the second experiment, we grew plants in high and low-light, and then monitored the behavior of tobacco hornworms on the stems of these plants in the laboratory. This experiment demonstrated that the light environment that tomato plants were grown in influenced the resting behavior of caterpillars. Furthermore, it demonstrated that both glandular and non-glandular trichomes impeded caterpillars from searching for food. Overall, this study indicated that plant resource availability can influence allocation to trichome defenses, and that these differences may affect insect herbivores.