Engineering of tomato type VI glandular trichomes for trans-chrysanthemic acid biosynthesis,the acid moiety of natural pyrethrin insecticides

Glandular trichomes, known as metabolic cell factories, have been proposed as highly suitable for metabolically engineering the production of plant high-value specialized metabolites. Natural pyrethrins, found only in Dalmatian pyrethrum (Tanacetum cinerariifolium), are insecticides with low mammalian toxicity and short environmental persistence. Type I pyrethrins are esters of the monoterpenoid trans-chrysanthemic acid with one of the three rethrolone-type alcohols. To test if glandular trichomes can be made to synthesize trans-chrysanthemic acid, we reconstructed its biosynthetic pathway in tomato type VI glandular trichomes, which produce large amounts of terpenoids that share the precursor dimethylallyl diphosphate (DMAPP) with this acid. This was achieved by coexpressing the trans-chrysanthemic acid pathway related genes including TcCDS encoding chrysanthemyl diphosphate synthase and the fusion gene of TcADH2 encoding the alcohol dehydrogenase 2 linked with TcALDH1 encoding the aldehyde dehydrogenase 1 under the control of a newly identified type VI glandular trichome-specific metallocarboxypeptidase inhibitor promoter. Whole tomato leaves harboring type VI glandular trichomes expressing all three aformentioned genes had a concentration of total trans-chrysanthemic acid that was about 1.5-fold higher (by mole number) than the levels of β-phellandrene, the dominant monoterpene present in non-transgenic leaves, while the levels of β-phellandrene and the representative sesquiterpene β-caryophyllene in transgenic leaves were reduced by 96% and 81%, respectively. These results suggest that the tomato type VI glandular trichome is an alternative platform for the biosynthesis of trans-chrysanthemic acid by metabolic engineering.     

ZFP5 encodes a functionally equivalent GIS protein to control trichome initiation

The Arabidopsis thaliana trichome development is a model system for understanding various aspects of plant cell development and differentiation. The C2H2 zinc finger proteins GIS, GIS2, and ZFP8 play important roles in controlling trichome initiation. In our recent study, we reported that a new C2H2 zinc finger protein, ZINC FINGER PROTEIN 5 (ZFP5), controls trichome cell development through GA signaling. ZFP5 acts upstream of GIS gene family and key trichome initiation regulators, and ZFP8 is the direct target gene of ZFP5. Here we show that ZFP5 encodes a protein functionally equivalent to GIS and GIS2 in controlling trichome initiation. Furthermore, similar to GIS2, ZFP5 is not involved in trichome branching.     

Accumulation and turnover of 2-tridecanone in Tetranychus urticae and its consequences for resistance of wild and cultivated tomatoes

In this study we assessed the dynamic changes of 2-tridecanone in a herbivorous mite (Tetranychus urticae) on tomato (Lycopersicon esculentum, cv. Moneymaker), a plant with methyl ketones in the tetracellular tips of the glandular trichomes (Type VI). We showed that spider mites accumulate 2-tridecanone when foraging on cultivated tomato. Thus, the rate of mite–trichome contact multiplied by the amount of toxin per trichome tip exceeded the relative rate of toxin turnover multiplied by the amount of toxin per mite. The relative rate of toxin turnover was estimated to be 1.1 per day on cucumber, a plant without this toxin. The amount per trichome tip varied from 0.33 ng for middle-leaf trichomes to 1.26 ng for main-stem trichomes. Hence, to achieve a static level of 2-tridecanone equal to 8–17 ng per mite – representing the level we found in mites on middle leaves – the rate of mite–trichome contact should be 26–57 per day. Because methyl ketone apparently accumulates in the spider mites on tomato, the rate of mite–trichome contact is probably higher than that. We expect the accumulation of ketones to occur especially on the stems of cultivated tomato, since this is the area most densely occupied with glandular hairs and because here the hairs have higher levels of the methyl ketones.Using dose–response relationships assessed earlier (Chatzivasileiadis and Sabelis, 1997, 1998), we estimated that the number of mite–trichome contacts causing 50% mortality per day is equal to 88 on a tomato stem, whereas it equals 70 for another strain of spider mites collected from cucumber. On wild tomato, L. hirsutum f. glabratum (PI 134417), just one to two contacts would suffice to cause 50% mortality per day. We suggest that methyl ketones from glandular hairs on tomato are an important mortality factor for spider mites on wild tomato and probably also on cultivated tomato.     

Herbivory-associated degradation of tomato trichomes and its impact on biological control of Aculops lycopersici

Tomato plants have their leaves, petioles and stems covered with glandular trichomes that protect the plant against two-spotted spider mites and many other herbivorous arthropods, but also hinder searching by phytoseiid mites and other natural enemies of these herbivores. This trichome cover creates competitor-free and enemy-free space for the tomato russet mite (TRM) Aculops lycopersici (Acari: Eriophyidae), being so minute that it can seek refuge and feed inbetween the glandular trichomes on tomato cultivars currently used in practice. Indeed, several species of predatory mites tested for biological control of TRM have been reported to feed and reproduce when offered TRM as prey in laboratory experiments, yet in practice these predator species appeared to be unable to prevent TRM outbreaks. Using the phytoseiid mite, Amblydromalus limonicus, we found exactly the same, but also obtained evidence for successful establishment of a population of this predatory mite on whole plants that had been previously infested with TRM. This successful establishment may be explained by our observation that the defensive barrier of glandular plant trichomes is literally dropped some time after TRM infestation of the tomato plants: the glandular trichome heads first rapidly develop a brownish discoloration after which they dry out and fall over onto the plant surface. Wherever TRM triggered this response, predatory mites were able to successfully establish a population. Nevertheless, biological control was still unsuccessful because trichome deterioration in TRM-infested areas takes a couple of days to take effect and because it is not a systemic response in the plant, thereby enabling TRM to seek temporary refuge from predation in pest-free trichome-dense areas which continue to be formed while the plant grows. We formulate a hypothesis unifying these observations into one framework with an explicit set of assumptions and predictions to be tested in future experiments.     

Purification and Characterization of Polyphenol Oxidase from Glandular Trichomes of Solanum berthaultii

Type A glandular trichomes of the wild potato (Solanum berthaultii Hawkes) entrap insects by rapidly polymerizing the trichome contents after breakage by insect contact. Polymerization of trichome exudate appears to be driven by a soluble polyphenol oxidase (PPO). PPO constitutes up to 70% of the protein in individually collected trichomes and reaches a concentration approaching 200 μm in these organs. Trichome PPO has been purified and shown to be a monomeric copper metalloprotein with an isoelectric point of 5.5, possessing only o-diphenol oxygen oxido-reductase activity, and is larger than most other reported PPOs, with relative molecular weight of 59,000. Chlorogenic and caffeic acid were the most readily oxidized of 14 phenolic substrates tested. Polyclonal antibodies raised against the relative molecular weight 59,000 S. berthaultii trichome PPO were used to show that S. tuberosum L. trichomes express low levels of a cross-reactive protein that lacks detectable PPO activity.     

Host plant effects on the behaviour and performance of Amblyseius swirskii (Acari: Phytoseiidae)

Biological control in ornamental crops is challenging due to the wide diversity of crops and cultivars. In this study, we tested the hypothesis that trichome density on different host plants influences the behavior and performance of the predatory mite Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae). Behavioural observations of this predator in the presence or absence of prey (western flower thrips, Frankliniella occidentalis Pergande) (Thysanoptera: Thripidae) were done on leaf squares of ornamental plant species differing in trichome density (rose, chrysanthemum and gerbera) and compared to a smooth surface (plastic). Tomato leaves were used to observe the influence of glandular trichomes. The performance of A. swirskii was assessed by measuring predation and oviposition rate. Behaviour of A. swirskii was influenced by plant species. Up to a certain density of trichomes, trichome number had a negative effect on walking speed. It was highest on plastic, followed by rose. No differences were found among chrysanthemum, gerbera and tomato. Walking speed was slightly higher on disks without prey. Proportion of time spent walking was the same on leaf disks of all plant species, with and without prey. No effect of glandular trichomes on tomato leaves was seen. Most thrips were killed and consumed on gerbera, and least on rose. Predation rates on chrysanthemum and plastic were intermediate. In contrast, no differences in oviposition rate were found among plant species. The results of this study indicate that trichome density can explain some of the variability in efficacy of A. swirskii on different crops. Release rates of A. swirskii may need to be adjusted depending on the crop in which it is used.     

TINY BRANCHED HAIR functions in multicellular trichome development through an ethylene pathway in Cucumis sativus L.

The fruit trichomes of Cucurbitaceae are widely desired in many Asian countries and have been a key determinant of cucumber (Cucumis sativus L.) cultivar selection for commercial production and breeding. However, our understanding of the initiation and development of cucumber trichomes is still limited. Here, we found that the cucumber TINY BRANCHED HAIR (TBH) gene is preferentially expressed in multicellular trichomes. Overexpression of CsTBH in tbh mutants restored the trichome phenotype and increased the percentage of female flowers, whereas silencing of CsTBH in wild-type plants resulted in stunted trichomes with a lower rate of female flowers. Furthermore, we provide evidence that CsTBH can directly bind to the promoters of cucumber 1-Aminocyclopropane-1-Carboxylate Synthase (CsACS) genes and regulate their expression, which affects multicellular trichome development, ethylene accumulation, and sex expression. Two cucumber acs mutants with different trichome morphology and sex morphs compared with their near-isogenic line further support our findings. Collectively, our study provides new information on the molecular mechanism of CsTBH in regulating multicellular trichome development and sex expression through an ethylene pathway.     

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.     

Glandular Trichomes ofCalceolaria adscendensLidl. (Scrophulariaceae): Histochemistry,Development and Ultrastructure

This paper reports the results of a study of the morphology and development of glandular trichomes in leaves ofCalceolaria adscendensLidl. using light and electron microscopy. Secretory trichomes started as outgrowths of epidermal cells; subsequent divisions gave rise to trichomes made up of a basal epidermal cell, a stalk cell and a two-celled secretory head. Ultrastructural characteristics of trichome cells were typical of terpene-producing structures. Previous phytochemical studies had revealed thatC. adscendensproduces diterpenes. Comparison withC. volckmanni,which produces triterpenes, and has trichomes with eight-celled secretory heads, suggests that there could be a relationship between the type of glandular trichome and the class of terpene produced. Further work is needed to test the hypothesis and to develop trichome characters as taxonomic tools.     

Identifying Three Ecological Chemotypes of Xanthium strumarium Glandular Trichomes Using a Combined NMR and LC-MS Method

Xanthanolides, as the sesquiterpene lactones, are reportedly the major components for the pharmacological properties of X. strumarium L. species. Phytochemical studies indicated that the glandular structures on the surface of plant tissues would form the primary sites for the accumulation of this class of the compounds. As the interface between plants and their natural enemies, glandular trichomes may vary with respect to which of their chemicals are sequestered against different herbivores in different ecologies. However, to date, no data are available on the chemical characterisation of X. strumarium glandular cells. In this study, the trichome secretions of the X. strumarium species originating from nineteen unique areas across eleven provinces in China, were analysed by HPLC, LC-ESI-MS and NMR. For the first time three distinct chemotypes of X. strumarium glandular trichomes were discovered along with the qualitative and quantitative evaluations of their presence of xanthanolides; these were designated glandular cell Types I, II, and III, respectively. The main xanthanolides in Type I cells were 8-epi-xanthatin and xanthumin while no xanthatin was detected. Xanthatin, 8-epi-xanthatin, and xanthumin dominated in Type II cells with comparable levels of each being present. For Type III cells, significantly higher concentrations of 8-epi-xanthatin or xanthinosin (relative to xanthatin) were detected with xanthinosin only being observed in this type. Further research will focus on understanding the ecological and molecular mechanism causing these chemotype differences in X. strumarium glandular structures.     

Zinc finger protein5 is required for the control of trichome initiation by acting upstream of zinc finger protein8 in Arabidopsis

Arabidopsis (Arabidopsis thaliana) trichome development is a model system for studying cell development, cell differentiation, and the cell cycle. Our previous studies have shown that the GLABROUS INFLORESCENCE STEMS (GIS) family genes, GIS, GIS2, and ZINC FINGER PROTEIN8 (ZFP8), control shoot maturation and epidermal cell fate by integrating gibberellins (GAs) and cytokinin signaling in Arabidopsis. Here, we show that a new C2H2 zinc finger protein, ZFP5, plays an important role in controlling trichome cell development through GA signaling. Overexpression of ZFP5 results in the formation of ectopic trichomes on carpels and other inflorescence organs. zfp5 loss-of-function mutants exhibit a reduced number of trichomes on sepals, cauline leaves, paraclades, and main inflorescence stems in comparison with wild-type plants. More importantly, it is found that ZFP5 mediates the regulation of trichome initiation by GAs. These results are consistent with ZFP5 expression patterns and the regional influence of GA on trichome initiation. The molecular analyses suggest that ZFP5 functions upstream of GIS, GIS2, ZFP8, and the key trichome initiation regulators GLABROUS1 (GL1) and GL3. Using a steroid-inducible activation of ZFP5 and chromatin immunoprecipitation experiments, we further demonstrate that ZFP8 is the direct target of ZFP5 in controlling epidermal cell differentiation.     

Trichomes as sensors: Detecting activity on the leaf surface

The dramatic movements of some carnivorous plants species are triggered by sensory structures derived from trichomes. While unusual plant species such as the Venus fly trap and sundews may be expected to have elaborate sensors to capture their insect prey, more modest plant species might not be expected to have similar sensory capabilities. Our recent work, however, has revealed that glandular trichomes on tomato (Solanum lycopersicum) appear to have a function similar to trigger hairs of carnivorous species, acting as “early warning” sensors. Using a combination of behavioral, molecular, and biochemical techniques, we determined that caterpillars, moths and mechanical disruption upregulate signaling molecules and defensive genes found in glandular trichomes. Importantly, we discovered that plants whose trichomes have been broken respond more vigorously when their defenses were induced. Taken together, our results suggest that glandular trichomes can act as sensors that detect activity on the leaf surface, and ready plants for herbivore attack.Key words: glandular trichome, induced responses, jasmonic acid, plant-insect interactions, sensor, Solanum lycopersicum, tomatoCertain plant species are renowned for their ability to respond to contact. The Venus fly trap (Dionaea muscipula) and sundew (Drosera) species come to mind quickly as obviously thigmotropic species. When an insect lands on these carnivorous plant species, dramatic movements ensue once the prey is detected. Some Drosera species respond to contact by bending their “tentacles” toward their trapped prey to further ensnare the victim and begin the process of digestion. These dramatic plant species have captured the attention of many scientists, including Darwin, who remarked on the “extraordinary sensitiveness of [their] glands to slight pressure” and surmised that the tentacles of sundew plants “existed primordially as glandular hairs.”¹ As is often the case, Darwin appears to have been quite right. Indeed, morphological and molecular work supports the notion that sundew tentacles and the trigger hairs of the Venus fly trap are homologous sensory structures likely derived from trichomes.^2,3Given Darwin’s appreciation of these trichome-derived sensory organs, he perhaps would not have been surprised by mounting evidence that suggests that trichomes may play even a broader sensory role for plants. We have recently found evidence that glandular trichomes can act as early detection sensors for some plant species.⁴ These trichomes can be disrupted by the footsteps of walking moths and caterpillars (and other forms of light touching), and this apparently minor plant damage leads to a state of defensive readiness that allows plants to respond to herbivory more quickly than undamaged plants. While this level of trichome-mediated detection does not result in the conspicuous responses of some carnivorous plant species, it still results in significant physiological changes that prepare plants for attack.In our recent effort, we worked with tomato (Solanum lycopersicum), using a combination of behavioral, molecular, and biochemical techniques to understand the role of trichomes in detecting activity on the leaf surface.⁴ Defense signaling has been well studied in tomato and there exists a variety of mutants whose defensive responses have been compromised. Moreover, it has been known that tomatoes have a variety of trichome types, including two types of glandular trichomes that burst upon contact with insects, releasing their cellular contents and physically impeding insects (Fig. 1).^5,6Open in a separate windowFigure 1Surface of a tomato leaf showing (A) intact rounded heads of glandular trichomes (black arrows) and (B) trichomes disrupted with a gloved hand (absence of rounded heads except for a few in the upper left corner [black arrows]). Images were captured at 36x magnification and were taken from different parts of the same leaf.To determine if plant defense pathways were induced by insect contact, we allowed three species of caterpillar (Manduca sexta, Heliothis virescens and Helicoverpa zea) and one species of moth (H. zea) to crawl over tomato leaves for ten minutes. As a positive control, we also lightly rubbed leaves with a gloved hand or a metal rod. Within time frames ranging from three to twenty-four hours all treatments, insect and otherwise, significantly induced defensive genes as measured by qRT-PCR. Using a combination of RT-PCR and in situ hybridization, we confirmed that JA-signaling and defensive genes are expressed in trichomes. A GC-MS-based technique then confirmed that JA was present in trichomes of undamaged plants and DAB staining, in combination with catalase treatment, provided evidence that hydrogren peroxide and JA are key signals mediating defensegene induction. These conclusions were further reinforced by experiments with def1 mutants, a line of tomato impaired in JA signaling, and accession LA3610, a tomato variety with reduced numbers of trichomes. Lastly, we conducted a factorial experiment both disrupting trichomes and treating tomato plants with methyl jasmonate (MeJA), which induces plant defenses and increases densities of trichomes.⁷ Results of this final experiment indicated that plants that received both treatments (i.e., MeJA and disruption) had greater defensive gene induction than plants that were only treated with MeJA or plants whose trichomes remained intact, suggesting that increases in trichomes may contribute to greater sensitivity to touch-induced responses.Taken together, our results are highly suggestive that trichomes can act as “early warning” detectors for plants. Moths seeking to lay eggs on tomato are likely to break trichomes as they explore leaves, upregulating plant defenses in anticipation of egg hatch and feeding by neonate caterpillars. Similarly, herbivores colonizing a new host plant and breaking trichomes on their way across a leaf also appear to “tip the plant off” to impending attack. Considering the drastic response of carnivorous plants to touch, perhaps it should not be surprising that trichomes can function more broadly as sensors. In an evolutionary context, it seems logical that trichomes took on this role. For many plant species, “hairy” varieties receive less herbivory,⁸ so within a population there could have been a fitness advantage in having more trichomes. Once established, this hairy phenotype could then have been refined via mutation and selection for trichome varieties that had functions adaptive for the plant, perhaps driving the evolution of glandular trichomes and their role as sensors.Granted, the generalized nature of our results would appear to indicate that plants could be “primed” by nearly any arthropod species that crosses one of their leaves. This would, of course, include natural enemies, which are capable of decreasing herbivore pressure and improving plant fitness.^9,10 However, it has been hypothesized that priming evolved due to high fitness costs associated with defensive induction following threats of only minor severity.¹¹ Priming provides an advantage by settling plants into an intermediate “ready” state that allows them to deploy strong defense responses more quickly and the fitness cost associated with being “primed” are lower than full defensive induction.¹² Presumably, fitness costs following priming due to natural enemyinduced trichome disruption would also be less than the cost incurred from a bout of unanticipated herbivory and, over the life of the plant, it would be worth the effort to prepare for attack even if the perceived risk is from a natural enemy and not a foe.Our results build on previously reported priming mechanisms that prepare plants for attack.^13,14 And they reveal an additional level of sophistication in the sensory capabilities of plants, which have already been shown to be able to detect nearby threats of herbivory and increase their defenses in response.^15,16 It seems that trichomes may have played a much wider role in shaping the nature of plant-animal interaction than previously recognized and we look forward to further work elaborating their function.     

Direct demonstration of duvatrienediol biosynthesis in glandular heads of tobacco trichomes

Biosynthesis of the diterpenes, α and β 4,8,13-duvatriene-1,3-diol, has been observed in detached, intact glandular heads from trichomes of Nicotiana tabacum, Tobacco Introduction 1068. This result shows directly that the glandular head portion of the trichome is capable of duvatrienediol biosynthesis. In additional experiments, all of the [¹⁴C] duvatrienediol formed from sodium [2-¹⁴C]acetate by leaf midrib sections was recovered with trichome exudate and surface washes. None was found in trichome stalk, epidermal or subepidermal tissue extracts. Also, removal of glandular heads and exudate from midrib sections reduced or eliminated duvatrienediol biosynthetic capacity. Together these results strongly suggest that glandular heads are the primary, and perhaps the only, site of duvatrienediol biosynthesis in this plant.

Incubation of detached, intact glandular heads with sodium [¹⁴C]acetate in the dark or incubation in the light in the presence of DCMU reduced incorporation into duvatrienediols by 97%. These results suggest that chloroplasts which are abundant in glandular heads are involved in the biogenesis of these compounds.

     

Hair,encoding a single C2H2 zinc‐finger protein,regulates multicellular trichome formation in tomato

Trichomes originate from the epidermal cells of nearly all terrestrial plants, which are specialized unicellular or multicellular structures. Although the molecular mechanism regulating unicellular trichome formation has been extensively characterized, most of the genes essential for multicellular trichome formation remain unknown. In this study, we identified an associated locus on the long arm of chromosome 10 using a genome‐wide association study (GWAS) on type‐I trichomes of 180 diverse Solanum lycopersicum (tomato) accessions. Using map‐based cloning we then cloned the key gene controlling the initiation of this type of trichome, named Hair (H), which encodes a single C2H2 zinc‐finger protein. Transgenic experiments showed that hair‐absent phenotype is caused by the deletion of the entire coding region of H. We identified three alleles of H containing several missense mutations and a nucleotide deletion, which result in amino acid substitutions and a reading frame shift, respectively. In addition, knockdown of H or Woolly (Wo) represses the formation of type‐I trichomes, suggesting that both regulators may function as a heterodimer. Direct protein–protein interaction between them was further detected through pull‐down and yeast two‐hybrid assays. In addition, ectopic expression of H in Nicotiana tabacum (tobacco) and expression of its homologs from Capsicum annuum (pepper) and tobacco in tomato can trigger trichome formation. Taken together, these findings suggest that the H gene may be functionally conserved in multicellular trichome formation in Solanaceae species.     

Isolation and Analysis of Differential Expressed ESTs from Stem Trichomes of Lycopersicon esculentum (Solanaceae)

Glandular trichomes are special organs involved in plant defense response and synthesis of volatile secondary metabolites, analyzing trichome specific expressed sequence tags will help us further understand the specific function of plant trichomes. In this paper, suppression subtractive hybridization（SSH） based on magnetic beads technology was used to isolate differential expressed genes of the glandular trichomes in Lycopersicon esculentum. The differential expressing cDNA library was constructed using the glandular trichomes cDNA as tester and the cDNA from the stem without glandular trichomes as driver. After randomly sequencing 108 differential ESTs, Blast2go program was used to do blastx, functional annotation and metabolism analysis. The results show that most ESTs are related to substance metabolism, response to stress, biotic or abiotic stimulus, and have binding and catalytic function. These differential genes lay the foundation for further research on defense mechanism of the tomato trichomes.