Characterization of two genes for the biosynthesis of the labdane diterpene Z-abienol in tobacco (Nicotiana tabacum) glandular trichomes

Leaves of tobacco (Nicotiana tabacum) are covered with glandular trichomes that produce sucrose esters and diterpenoids in varying quantities, depending on cultivar type. The bicyclic diterpene Z‐abienol is the major labdanoid present in some oriental tobacco cultivars, where it constitutes a precursor of important flavours and aromas. We describe here the identification and characterization of two genes governing the biosynthesis of Z‐abienol in N. tabacum. As for other angiosperm labdanoid diterpenes, the biosynthesis of Z‐abienol proceeds in two steps. NtCPS2 encodes a class‐II terpene synthase that synthesizes 8‐hydroxy‐copalyl diphosphate, and NtABS encodes a kaurene synthase‐like (KSL) protein that uses 8‐hydroxy‐copalyl diphosphate to produce Z‐abienol. Phylogenetic analysis indicates that NtABS belongs to a distinct clade of KSL proteins that comprises the recently identified tomato (Solanum habrochaites) santalene and bergamotene synthase. RT‐PCR results show that both genes are preferentially expressed in trichomes. Moreover, microscopy of NtCPS2 promoter‐GUS fusion transgenics demonstrated a high specificity of expression to trichome glandular cells. Ectopic expression of both genes, but not of either one alone, driven by a trichome‐specific promoter in transgenic Nicotiana sylvestris conferred Z‐abienol formation to this species, which does not normally produce it. Furthermore, sequence analysis of over 100 tobacco cultivars revealed polymorphisms in NtCPS2 that lead to a prematurely truncated protein in cultivars lacking Z‐abienol, thus establishing NtCPS2 as a major gene controlling Z‐abienol biosynthesis in tobacco. These results offer new perspectives for tobacco breeding and the metabolic engineering of labdanoid diterpenes, as well as for structure–function relationship studies of terpene synthases.     

Evolution of oil-producing trichomes in Sisyrinchium (Iridaceae): insights from the first comprehensive phylogenetic analysis of the genus

Background and Aims

Sisyrinchium (Iridaceae: Iridoideae: Sisyrinchieae) is one of the largest, most widespread and most taxonomically complex genera in Iridaceae, with all species except one native to the American continent. Phylogenetic relationships within the genus were investigated and the evolution of oil-producing structures related to specialized oil-bee pollination examined.

Methods

Phylogenetic analyses based on eight molecular markers obtained from 101 Sisyrinchium accessions representing 85 species were conducted in the first extensive phylogenetic analysis of the genus. Total evidence analyses confirmed the monophyly of the genus and retrieved nine major clades weakly connected to the subdivisions previously recognized. The resulting phylogenetic hypothesis was used to reconstruct biogeographical patterns, and to trace the evolutionary origin of glandular trichomes present in the flowers of several species.

Key Results and Conclusions

Glandular trichomes evolved three times independently in the genus. In two cases, these glandular trichomes are oil-secreting, suggesting that the corresponding flowers might be pollinated by oil-bees. Biogeographical patterns indicate expansions from Central America and the northern Andes to the subandean ranges between Chile and Argentina and to the extended area of the Paraná river basin. The distribution of oil-flower species across the phylogenetic trees suggests that oil-producing trichomes may have played a key role in the diversification of the genus, a hypothesis that requires future testing.     

Comparative anatomy of the nectary spur in selected species of Aeridinae (Orchidaceae)

Background and Aims

To date, the structure of the nectary spur of Aeridinae has not been studied in detail, and data relating to the nectaries of ornithophilous orchids remain scarce. The present paper compares the structural organization of the floral nectary in a range of Aeridinae species, including both entomophilous and ornithophilous taxa.

Methods

Nectary spurs of Ascocentrum ampullaceum (Roxb.) Schltr. var. aurantiacum Pradhan, A. curvifolium (Lindl.) Schltr., A. garayi Christenson, Papilionanthe vandarum (Rchb.f.) Garay, Schoenorchis gemmata (Lindl.) J.J. Sm., Sedirea japonica (Rchb.f.) Garay & H.R. Sweet and Stereochilus dalatensis (Guillaumin) Garay were examined by means of light microscopy, scanning electron microscopy and transmission electron microscopy.

Key Results and Conclusions

The diverse anatomy of the nectary is described for a range of Aeridinae species. All species of Ascocentrum investigated displayed features characteristic of ornithophilous taxa. They have weakly zygomorphic, scentless, red or orange flowers, display diurnal anthesis, possess cryptic anther caps and produce nectar that is secluded in a relatively massive nectary spur. Unicellular, secretory hairs line the lumen at the middle part of the spur. Generally, however, with the exception of Papilionanthe vandarum, the nectary spurs of all entomophilous species studied here (Schoenorchis gemmata, Sedirea japonica, Stereochilus dalatensis) lack secretory trichomes. Moreover, collenchymatous secretory tissue, present only in the nectary spur of Asiatic Ascocentrum species, closely resembles that found in nectaries of certain Neotropical species that are hummingbird-pollinated and assigned to subtribes Maxillariinae Benth., Laeliinae Benth. and Oncidiinae Benth. This similarity in anatomical organization of the nectary, regardless of geographical distribution and phylogeny, indicates convergence.     

Comparative labellar micromorphology of Zygopetalinae (Orchidaceae)

Background and Aims

Molecular evidence indicates that the Neotropical sub-tribe Zygopetalinae is sister to Maxillariinae. Most members of the latter sub-tribe have deceit pollination strategies, but some species produce rewards such as nectar, pseudopollen, resin and wax, and are pollinated by a range of pollinators that include stingless bees (Meliponini), wasps and hummingbirds. By contrast, relatively little is known about the pollination of Zygopetalinae species. However, some are pollinated by fragrance-gathering, male euglossine bees or employ nectar deceit strategies. The aim of this study is to describe the labellar micromorphology of Zygopetalinae and to compare it with that of Maxillariinae sensu lato (s.l.) as part of an ongoing project to record the range of labellar characters found within the tribe Maxillarieae, and to assess whether these characters represent synapomorphies or homoplasies resulting from similar pollination pressures.

Methods

The labella of 31 species of Zygopetalinae, including Cryptarrhena R. Br. and representatives of the Zygopetalum, Huntleya and Warrea clades, were examined using light microscopy and scanning electron microscopy, and the range of labellar characters was recorded. These characters were subsequently compared with those of Maxillariinae s.l. which formed the subject of our previous investigations.

Key Results and Conclusions

The labellar micromorphology of Zygopetalinae is less diverse than that of Maxillariinae and does not reflect the currently accepted phylogeny of the former sub-tribe based on molecular studies. Instead, the relative uniformity in labellar micromorphology of Zygopetalinae is probably due to homoplasies resulting from similar pollinator pressures. Labellar trichomes are relatively uncommon in Zygopetalinae, but occur in certain members of both the Zygopetalum and Huntleya clades. Trichomes are unbranched, uniseriate and multicellular with rounded apices, or unbranched and unicellular, with tapering, pointed and flexuose apices. Hitherto, unicellular trichomes of this kind have been observed only for euglossophilous orchid taxa, and the adoption of a relatively limited range of pollination strategies by Zygopetalinae may have resulted in reduced investment in micromorphological labellar characters.     

A Cytochemical Study of ACPase and ALPase Activity in the Laminar Hair Cells of Bresenia shreberi

The ultrastructural distribution of acid phosphatase (ACPase) alkaline phosphatase (ALPase) and the changes of their activities in the laminar hair cells of Bresenia shreberi Geml. were studied by means of lead phosphate preciptation method during their development. The ACPase activity became detactable at the stage of mucilage secretion of the head cell, which was located at endoplasmic reticulum (Er),cuticle (Cu) and tonoplast (Tp) of the head cell, and nuclear membrane (NM), plasmodesmata (P1) and wall ingrowths (WI) of the stalk cell, and WI and plasmolemma of basal cell. ALPase activity showed its first appearance at the mitochondria(Mi) of the basal cell at the stage of head cell formation. At the stage of head cell secretion, ALPase activity was located at Er, Cu, Mi of head cell, and NM, Go, Er, WI and P1 of stalk cell. ALPase activity at Mi of basal cell was most intensive. The results indicated that ACPase and ALPase possessed the function of promoting cell differentiation and material transfer in the process of hair cell development and that the material for synthesizing mucilage in head cell was provided by the basal cells and the parenchyma cells nearby.     

Leaf hairs influence searching efficiency and predation rate of the predatory mite Phytoseiulus persimilis (Acari: Phytoseiidae)

The effect of leaf hairs on searching efficiency of adult female Phytoseiulus persimilis was investigated. For this purpose we used the ornamental crop Gerbera jamesonii and determined the predator's searching efficiency on three cultivars that differ largely in the density of leaf hairs on the undersurface of the leaves. Walking speed of the mites was highest on the cultivar with the lowest leaf hair density. Walking activity, defined as the percentage of time spent walking, was not dependent on leaf hair density of the cultivars. At both prey densities tested, time until first predation increased with leaf hair density. The predation rate of adult female P. persimilis is affected by trichome density, particularly when prey density is low. At prey densities of 1.3 and 2.5 Tetranychus urticae eggs cm–2, predation rate was inversely related to leaf hair density. At a prey density of 8.0 eggs cm–2 no significant effect of leaf hair density on predation rate was found. These negative effects on searching efficiency and predation success at low prey density of P. persimilis suggest that biological control of T. urticae on gerbera may be hampered by leaf hairs. © Rapid Science Ltd. 1998     

Effect of plant variety, plant age and photoperiod on glandular pubescence and host-plant resistance to potato moth (Phthorimaea operculella) in Lycopersicon spp.

The effect of plant age and daylength on glandular pubescence was determined for two lines of tomato derived from Lycopersicon hirsutum (BTN 979 and LA 1777A) and a variety of L. esculentum (N 91‐1‐1‐1‐1). Densities of type I, IV, VI and VII glandular trichomes were lowest in N 91‐1‐1‐1‐1 and, over all varieties, were more dense on plants aged greater than 6 wk. Daylength interacted with variety to significantly affect densities of type VII trichomes only. Host‐plant resistance to Phthorimaea operculella was determined in preliminary tests using insects cultured from founders from a potato crop and in confirmatory tests using (less readily available) insects recovered from foliage of a tomato crop. Mortality of ex‐potato neonates on LA 1777 A and BTN 979 foliage was higher 18 h after placement than for N 91‐1‐1‐1‐1, with no effect of day length or plant age. Mortality for ex‐tomato neonates followed a similar trend. Ten days later, two‐thirds of ex‐tomato larvae had established mines on N 91‐1‐1‐1‐1 but fewer (16.7%) were live on other varieties. Stepwise multiple regression using variety as the sole factor was significant (P<0.001) in accounting for 61.4% of the variation in ex‐tomato larval survival but addition of other factors to the regression model was not significant. BTN 979 supported fewer, smaller adults to develop than did N 91‐1‐1‐1‐1, whilst no adults developed on LA 1777 A. In a non‐choice test using ex‐potato adults, significantly more eggs were laid on N 91‐1‐1‐1‐1 than on L. hirsutum varieties and 9‐wk‐old plants were preferred over plants three weeks older or younger. The same variety and plant age trends were evident in a free‐choice test using ex‐tomato adults.     

Leaf anatomy of Gluta (L.) Ding Hou (Anacardiaceae)

An anatomical study of the leaves of 21 species of Gluta (L.) Ding Hou (Anacardiaceae) reveals two major groups of species which reflect the original groups of Gluta L. and Melanorrhoea Wall., and a smaller group showing intermediate, or an admixture of, characters. The anatomical characters found to be of most use in this respect are: stomatal outline in surface view; stomatal density; glandular trichomes present/absent; glandular trichome body raised/sunken; cuticle striate/not striate; midrib dimensions as seen in transverse section; kind of simple trichomes (trichome-types 1–4); epidermal cell anticlinal wall undulation and whether visible or not on cuticular surface; resin ducts present/absent in medullary parenchyma of midrib. These characters have been used in a key to the species. Some evidence is given that the lacquer covering the leaf surface of some species is produced by the terminal cells of the glandular trichomes.     

Accessory Pollen Adhesive from Glandular Trichomes on the Anthers of Leonurus sibiricus L. (Lamiaceae)

Abstract: Glandular trichomes (ca. 16 per anther) on the anthers of Leonurus sibiricus produce a secretion that, when touched, is liberated at once and becomes sticky when in contact with the air. With successive visitations of the pollinators (species of Bombus in naturalized populations) the number of secretion‐containing glands on each anther diminishes by mechanical rupture. On the pollinators, the secretion mixed with pollen was found adhered to the integument on the parts making contact with the anthers and stigma, mostly on the scape of the antennae. These trichomes are anatomically identical to the glandular scales common in the entire family and are formed by a multicellular cuticle‐bounded structure, with a foot and head. The secretion is accumulated as a milky emulsion under the cuticle, outside the primary cell wall, and is liberated by rupture of the cuticle. The composition possibly differs from what generally distinguishes these glandular trichomes, i.e. volatile oils that give these plants their particular smell. Such volatile compounds are generally assumed to have defensive or attractive functions, different from those observed in this study, which would be strictly mechanical.     

Atypical pseudopollen-forming hairs in Maxillaria (Orchidaceae)

Pseudopollen is found on the labella of several species of Maxillaria and is formed by the fragmentation of uniseriate, multicellular, moniliform trichomes. The resultant component cells are rich in protein. Since flowers of pseudopollen-forming species generally lack nectar, it is probable that pseudopollen gradually replaced nectar as the pollinator reward. However, direct evidence for this hypothesis is lacking. The present paper examines the labellar micromorphology of five Brazilian species of Maxillaria assigned to the M. discolor alliance. The flowers of two of these species, namely M. violaceopunctata and M. villosa , produce both food hairs and a lipoidal, labellar secretion which is rich in aromatic amino acids. Moreover, in the case of M. violaceopunctata , the secretion was found to contain reducing sugars. This may represent an intermediate stage in the transition from nectar-producing to pseudopollen-forming flowers. SEM studies indicate how pseudopollen-forming, moniliform trichomes, such as those found in the Maxillaria grandiflora complex, may have evolved from simple, uniseriate, multicellular hairs.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 143 , 151−158.