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Plant Glandular Trichomes: Natural Cell Factories of High Biotechnological Interest
Abstract
Multicellular glandular trichomes are epidermal outgrowths characterized by the presence of a head made of cells that have the ability to secrete or store large quantities of specialized metabolites. Our understanding of the transcriptional control of glandular trichome initiation and development is still in its infancy. This review points to some central questions that need to be addressed to better understand how such specialized cell structures arise from the plant protodermis. A key and unique feature of glandular trichomes is their ability to synthesize and secrete large amounts, relative to their size, of a limited number of metabolites. As such, they qualify as true cell factories, making them interesting targets for metabolic engineering. In this review, recent advances regarding terpene metabolic engineering are highlighted, with a special focus on tobacco (Nicotiana tabacum). In particular, the choice of transcriptional promoters to drive transgene expression and the best ways to sink existing pools of terpene precursors are discussed. The bioavailability of existing pools of natural precursor molecules is a key parameter and is controlled by so-called cross talk between different biosynthetic pathways. As highlighted in this review, the exact nature and extent of such cross talk are only partially understood at present. In the future, awareness of, and detailed knowledge on, the biology of plant glandular trichome development and metabolism will generate new leads to tap the largely unexploited potential of glandular trichomes in plant resistance to pests and lead to the improved production of specialized metabolites with high industrial or pharmacological value.
... overexpressing the peppermint geranyl diphosphate synthase small subunit in tobacco. Other deleterious 517 effects were observed for different terpenoids, including chlorosis, dwarfism, and a reduction in fertility 518 (Huchelmann et al., 2017). Because of the similarity of the phenotypes observed in different reports, 519 cytotoxicity of the new metabolites is considered not to be the only cause, and perhaps plant depletion of its 520 essential terpenoid precursors (IPP/DMAPP) is also playing a role. ...
... It is The copyright holder for this preprint this version posted August 3, 2023. ; https://doi.org/10.1101/2023.08.02.551635 doi: bioRxiv preprint 15 include accumulation in trichomes using specific promoters and transporters (Huchelmann et al., 2017). This 542 might not represent an ideal solution for the accumulation of volatile compounds in tobacco, since it does 543 not possess peltate trichomes such as those of aromatic plants, but rather, its capitate trichomes are 544 specialized for the secretion on the leaf surface of non-volatile diterpenes and phytoalexins (Tissier et al.,545 2017). ...
Irregular monoterpenes are important precursors of different compounds employed in pest control such as insecticides and insect sex pheromones. Metabolically engineered plants are appealing as biofactories of such compounds, but specially as potential live biodispensers of related bioactive volatiles, which could be continuously emitted to the environment from different plant tissues. Here we assess the use of cultivated tobacco and Nicotiana benthamiana as biofactories for the irregular monoterpenes chrysanthemol and lavandulol. We evaluate the impact of high levels of constitutive metabolite production on the plant physiology and biomass, and their biosynthetic dynamics for different plant tissues and developmental stages. As an example of an active pheromone compound, we super-transformed the best lavandulol-producing tobacco line with an acetyl transferase gene to obtain a tobacco lavandulyl acetate biodispenser emitting up to 0.63 mg of lavandulyl acetate per plant every day. We estimate that with these volatile emission levels, between 200 and 500 plants per hectare would be sufficient to ensure a daily emission of pheromones comparable to commercial lures. This is an important step towards plant-based sustainable solutions for pest control, and it lays the ground for further developing biofactories for other irregular monoterpenoid pheromones, whose biosynthetic genes are yet unknown.
... Non-glandular trichomes can strengthen the role of resistance in abiotic stress by promoting normal plant growth, under condition of extreme high or low temperature, drought and UV irradiation. Glandular trichomes can produce specialized metabolites that improve plant fitness for the environment [33]. The glandular trichomes can contribute to the accumulation and secretion of some alkaloids to resist insects, such as nicotine and terpenoid alkaloids [34]. ...
Foliar micromorphological and anatomical characteristics of Pogostemon heyneanus Benth. (Lamiaceae) was investigated in order to describe its comprehensive characterization and its association with the presence of essential oils. The methods used in this study involved several methods such as cross-section using a sliding microtome and epidermal peeling that were observed under light microscope, and foliar micromorphological characteristics that were observed under scanning electron microscope (SEM). Results showed leaf anatomical and micromorphological characteristics that could be useful in species identification and the localization of chemical properties. The leaf epidermal surfaces were characterized by curved to sinuous anticlinal walls (adaxial side) and sinuous anticlinal walls (abaxial side). The diacytic and anisocytic types of stomata were present only on the abaxial surface. The features of the stem is quadrangular and the well-developed collenchyma function. The sclerenchyma cells are present as clusters at the outer layer of vascular bundles and continuously surround the vascular tissue. Then, there were three forms of crystals found, namely star shaped crystals, prismatic crystals and raphides in the pith area. Eight types of trichomes were observed: simple unicellular, simple multicellular, peltate, short-stalked capitate (unicellular head), short-stalked capitate (bicellular head), short-swollen multistalked (unicellular acute head), long-stalked capitate, and long-swollen stalked capitate (disk head) trichomes. The presence of various glandular trichomes on the leaf surfaces may serve as secretory sites where secondary metabolites or essential oils are produced. The findings on the foliar and stem anatomical and micromorphological characteristics are very useful for the medicinal herbs industry as well as being of taxonomic value.
... Moreover, sequestration and storage of newly produced metabolites seems to be a crucial aspect of high-level metabolite formation in host organisms. Especially glandular trichomes fulfill the role of specialized organs optimized to produce and store extreme amounts of natural products incompatible with the aqueous environment within a cell (Huchelmann et al. 2017). ...
Results: The reporter gene GFP::licBM3 as well as three biosynthetic genes leading to the formation of the cannabinoid precursor olivetolic acid were adopted to the modular cloning standard GoldenBraid, transiently expressed in two chemotypes of N. cataria and compared to Nicotiana benthamiana. To estimate the expression efficiency in both hosts, quantification of the reporter activity was carried out with a sensitive and specific lichenase assay. While N. benthamiana exhibited lichenase activity of 676 +/- 94 umol g-1 s-1 (Gerasimenko et al. 2019), N. cataria cultivar '1000', and the cultivar 'Citriodora' showed an activity of 37 +/- 8 umol g-1 s-1 and 18 +/-4 umol g-1 s-1, respectively. Further, combinatorial expression of genes involved in cannabinoid biosynthetic pathway acyl-activating enzyme 1 (aae1), olivetol synthase (ols) and olivetolic acid cyclase (oac) in N. cataria cv. resulted presumably in the in vivo production of olivetolic acid glycosides.
Conclusion: Nepeta cataria is amenable to Agrobacterium-mediated transient expression and could serve as a novel chassis for the engineering of secondary metabolic pathways and transient evaluation of heterologous genes.
... While the genetic and morphological underpinning of cannabinoid accumulation have been well studied (Schilling et al. 2021), the role and function of cannabinoids in the physiology of the plant itself is yet to be determined. Approximately 30% of all vascular plant species have glandular trichomes to secrete and store secondary metabolites, including flavonoids, monoterpenes, or sesquiterpene lactones (Huchelmann et al. 2017). The contribution to many of these compounds in defense against diverse abiotic and biotic environmental challenges have been clearly shown and are reviewed in detail elsewhere (Peiffer et al. 2009;Agati et al. 2012;Schuurink and Tissier 2020). ...
i>Cannabis sativa is one of the oldest cultivated crops, used for its fiber and medicinal properties. The cannabis plant synthesizes a myriad of secondary metabolites, but the most valuable products from a medical and commercial standpoint are cannabinoids. Despite significant advances in elucidating the biochemistry and genetics that govern cannabinoid accumulation, we still do not have conclusive evidence for the role of these secondary metabolites in the physiology of C. sativa . In line with known functions of other secondary metabolites, the protective functions of cannabinoids against temperature stress, poor micronutrient soil content, drought, UV-B radiation, and as anti-microbial agents have been suggested, but are yet to be conclusively demonstrated. Recent research suggests that the environment has a major effect on cannabis growth and productivity, but the relationship between stress, cannabinoid accumulation, and plant health is complex. Here, we summarize the current insights on how abiotic and biotic stress affect C. sativa biology. We also examine the available evidence to support the hypothesis for the protective function of cannabinoids against environmental stressors. Maintaining optimal growth and high cannabinoid synthesis is a balancing act, one that can only be achieved by better understanding of the effects on the environment on the cannabis plant.
... In the OS-1 genome, we identified the genes involved in the metabolism of acyl sugar, monocyclic and acyclic monoterpenes, and carbohydrate esterase family I. Acyl sugars with long-chain esters are involved in the insecticidal activity (Fan et al., 2019), whereas carbohydrate esterase family-I is involved in the removal of esters from the carbohydrate core (Nakamura et al., 2017). Terpenoids are associated with antimicrobial activity and bacteria can metabolize them for further use as carbon sources (Huchelmann et al., 2017;Schuurink and Tissier, 2020). The ability of nitrogen metabolism was evidenced by the presence of genes involved in assimilatory and dissimilatory nitrate reduction (ANR and DNR) (Huang et al., 2020), glutamate metabolism (Paul et al., 2007) and cyanide metabolism, respectively (Lin et al., 2017). ...
A metal-resistant bacterium Pseudomonas parafulva OS-1 was isolated from waste-contaminated soil in Ranchi City, India. The isolated strain OS-1 showed its growth at 25–45°C, pH 5.0–9.0, and in the presence of ZnSO 4 (upto 5 mM). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain OS-1 belonged to the genus Pseudomonas and was most closely related to parafulva species. To unravel the genomic features, we sequenced the complete genome of P. parafulva OS-1 using Illumina HiSeq 4,000 sequencing platform. The results of average nucleotide identity (ANI) analysis indicated the closest similarity of OS-1 to P. parafulva PRS09-11288 and P. parafulva DTSP2. The metabolic potential of P. parafulva OS-1 based on Clusters of Othologous Genes (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated a high number of genes related to stress protection, metal resistance, and multiple drug-efflux, etc., which is relatively rare in P. parafulva strains. Compared with other parafulva strains, P. parafulva OS-1 was found to have the unique β-lactam resistance and type VI secretion system (T6SS) gene. Additionally, its genomes encode various CAZymes such as glycoside hydrolases and other genes associated with lignocellulose breakdown, suggesting that strain OS-1 have strong biomass degradation potential. The presence of genomic complexity in the OS-1 genome indicates that horizontal gene transfer (HGT) might happen during evolution. Therefore, genomic and comparative genome analysis of parafulva strains is valuable for further understanding the mechanism of resistance to metal stress and opens a perspective to exploit a newly isolated bacterium for biotechnological applications.
Species from genus Artemisia are widely distributed throughout temperate regions of the northern hemisphere and many cultures have a long-standing traditional use of these plants as herbal remedies, liquors, cosmetics, spices, etc. Nowadays, the discovery of new plant-derived products to be used as food supplements or drugs has been pushed by the exploitation of bioprospection approaches. Often driven by the knowledge derived from the ethnobotanical use of plants, bioprospection explores the existing biodiversity through integration of modern omics techniques with targeted bioactivity assays.
In this work we set up a bioprospection plan to investigate the phytochemical diversity and the potential bioactivity of five Artemisiaspecies with recognized ethnobotanical tradition (A. absinthium, A. alba, A. annua, A. verlotiorum and A. vulgaris), growing wild in the natural areas of the Verona province. We characterized the specialized metabolomes of the species (including sesquiterpenoids from the artemisinin biosynthesis pathway) through an LC-MS based untargeted approach and, in order to identify potential bioactive metabolites, we correlated their composition with the in vitro antioxidant activity. We propose as potential bioactive compounds several isomers of caffeoyl and feruloyl quinic acid esters (e.g. dicaffeoylquinic acids, feruloylquinic acids and caffeoylferuloylquinic acids), which strongly characterize the most antioxidant species A. verlotiorum and A. annua. Morevoer, in this study we report for the first time the occurrence of sesquiterpenoids from the artemisinin biosynthesis pathway in the species A. alba.
Gossypol and the related terpenoids are stored in the pigment gland to protect cotton plants from biotic stresses, but little is known about the synthetic sites of these metabolites.
Here, we showed that GoPGF , a key gene regulating gland formation, was expressed in gland cells and roots. The chromatin immunoprecipitation sequencing (ChIP‐seq) analysis demonstrated that GoPGF targets GhJUB1 to regulate gland morphogenesis.
RNA‐sequencing (RNA‐seq) showed high accumulation of gossypol biosynthetic genes in gland cells. Moreover, integrated analysis of the ChIP‐seq and RNA‐seq data revealed that GoPGF binds to the promoter of several gossypol biosynthetic genes. The cotton callus overexpressing GoPGF had dramatically increased the gossypol levels, indicating that GoPGF can directly activate the biosynthesis of gossypol. In addition, the gopgf mutant analysis revealed the existence of both GoPGF‐dependent and ‐independent regulation of gossypol production in cotton roots.
Our study revealed that the pigment glands are synthetic sites of gossypol in aerial parts of cotton and that GoPGF plays a dual role in regulating gland morphogenesis and gossypol biosynthesis. The study provides new insights for exploring the complex relationship between glands and the metabolites they store in cotton and other plant species.
Is Cannabis a boon or bane? Cannabis sativa has long been a versatile crop for fiber extraction (industrial hemp), traditional Chinese medicine (hemp seeds), and recreational drugs (marijuana). Cannabis faced global prohibition in the 20th century because of the psychoactive properties of ∆9-tetrahydrocannabinol; however, recently, the perspective has changed with the recognition of additional therapeutic values, particularly the pharmacological potential of cannabidiol. A comprehensive understanding of underlying mechanism of cannabinoid biosynthesis is necessary to cultivate and promote globally the medicinal application of Cannabis resources. Here, we comprehensively review the historical usage of Cannabis, biosynthesis of trichome-specific cannabinoids, regulatory network of trichome development, and synthetic biology of cannabinoids. This review provides valuable insights into the efficient biosynthesis and green production of cannabinoids, and the development and utilization of novel Cannabis varieties.
Plant growth and development are controlled by a complex gene regulatory network, which is currently a focal point of research. It has been established that epigenetic factors play a crucial role in plant growth. Trichomes, specialized appendages that arise from epidermal cells, are of great significance in plant growth and development. As a model system for studying plant development, trichomes possess both commercial and research value. Epigenetic regulation have only recently been implicated in the development of trichomes in a limited number of studies, and microRNA-mediated post-transcriptional regulation appears to dominate in this context. In light of this, we have conducted a review that explores the interplay between epigenetic regulations and the formation of plant trichomes, building upon existing knowledge of hormones and trascription factors in trichome development. Through this review, we aim to deepen our understanding of the regulatory mechanisms underlying trichome formation and shed light on future avenues of research in the field of epigenetics as it pertains to epidermal hair growth.
Background
Trichomes, developing from the epidermis of nearly all terrestrial plants, provide good structural resistance against insect herbivores and an excellent model for studying the molecular mechanisms underlying cell fate determination. Regulation of trichomes in Rosids has been well characterized. However, little is known about the cell proliferation molecular processes during multicellular trichome formation in Asterids.
Results
In this study, we identified two point mutations in a novel allele (Wov) at Wo locus. Ectopic expression of Wov in tobacco and potato induces much more trichome formation than wild type. To gain new insights into the underlying mechanisms during the processes of these trichomes formation, we compared the gene expression profiles between Wov transgenic and wild-type tobacco by RNA-seq analysis. A total of 544 co-DEGs were detected between transgenic and wild-type tobacco. Functional assignments of the co-DEGs indicated that 33 reliable pathways are altered in transgenic tobacco plants. The most noticeable pathways are fatty acid metabolism, amino acid biosynthesis and metabolism, and plant hormone signal transduction. Results suggest that these enhanced processes are critical for the cell proliferation during multicellular trichome formation in transgenic plants. In addition, the transcriptional levels of homologues of trichome regulators in Rosids were not significantly changed, whereas homologues of genes (Wo and SlCycB2) in Asterids were significantly upregulated in Wov transgenic tobacco plants.
Conclusions
This study presents a global picture of the gene expression changes induced by Wov- gene in tobacco. And the results provided us new insight into the molecular processes controlling multicellular formation in tobacco. Furthermore, we inferred that trichomes in solanaceous species might share a common network.
Background
Tobacco (Nicotiana tabacum) is an important plant model system that has played a key role in the early development of molecular plant biology. The tobacco genome is large and its characterisation challenging because it is an allotetraploid, likely arising from hybridisation between diploid N. sylvestris and N. tomentosiformis ancestors. A draft assembly was recently published for N. tabacum, but because of the aforementioned genome complexities it was of limited utility due to a high level of fragmentation.
Results
Here we report an improved tobacco genome assembly, which, aided by the application of optical mapping, achieves an N50 size of 2.17 Mb and enables anchoring of 64% of the genome to pseudomolecules; a significant increase from the previous value of 19%. We use this assembly to identify two homeologous genes that explain the differentiation of the burley tobacco market class, with potential for greater understanding of Nitrogen Utilization Efficiency and Nitrogen Use Efficiency in plants; an important trait for future sustainability of agricultural production.
Conclusions
Development of an improved genome assembly for N. tabacum enables what we believe to be the first successful map-based gene discovery for the species, and demonstrates the value of an improved assembly for future research in this model and commercially-important species.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-017-3791-6) contains supplementary material, which is available to authorized users.
Artemisinin, a sesquiterpenoid endoperoxide, isolated from the plant Artemisia annua L., is widely used in the treatment of malaria. Another sesquiterpenoid, β-caryophyllene having antibiotic, antioxidant, anticarcinogenic and local anesthetic activities, is also presented in A. annua. The role played by sesquiterpene transporters in trichomes and accumulation of these metabolites is poorly understood in A. annua and in trichomes of other plant species. We identified AaPDR3, encoding a pleiotropic drug resistance (PDR) transporter located to the plasma membrane from A. annua. Expression of AaPDR3 is tissue-specifically and developmentally regulated in A. annua. GUS activity is primarily restricted to T-shaped trichomes of old leaves and roots of transgenic A. annua plants expressing proAaPDR3: GUS. The level of β-caryophyllene was decreased in transgenic A. annua plants expressing AaPDR3-RNAi while transgenic A. annua plants expressing increased levels of AaPDR3 accumulated higher levels of β-caryophyllene. When AaPDR3 was expressed in transformed yeast, yeasts expressing AaPDR3 accumulated more β-caryophyllene, rather than germacrene D and β-farnesene, compared to the non-expressing control.
The co-operation of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, operating in parallel in plants to generate isoprenoid precursors, has been studied extensively. Elucidation of the isoprenoid metabolic pathways is indispensable for rational design of plant and microbial systems for production of industrially valuable terpenoids. Here we describe a new method, based on numerical modeling of mass spectra of metabolically labeled dolichols (Dols), designed to quantitatively follow the co-operation of the MVA and MEP reprogrammed upon osmotic stress (sorbitol treatment). The contribution of the MEP pathway increased significantly (reaching 100%) exclusively for the dominating Dols while for long-chain Dols the relative input of the MEP and MVA pathways remained unchanged suggesting divergent site of synthesis of dominating and long-chain Dols. Analysis of numerically modeled dolichol mass spectra is a novel method to follow modulation of the concomitant activity of isoprenoid generating pathways in plant cells, and additionally, it suggests exchange of isoprenoid intermediates between plastids and peroxisomes.
Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is the most abundant enzyme in plants and is responsible for CO2 fixation during photosynthesis. This enzyme is assembled from eight large subunits (RbcL) encoded by a single chloroplast gene, and eight small subunits (RbcS) encoded by a nuclear gene family. Rubisco is primarily found in the chloroplasts of mesophyll (C3 plants), bundle-sheath (C4 plants) and guard cells. In certain species, photosynthesis also takes place in the secretory cells of glandular trichomes, which are epidermal outgrowths (hairs) involved in the secretion of specialized metabolites. However photosynthesis and, in particular, Rubisco have not been characterized in trichomes. Here, we show that Nicotiana tabacum trichomes contain a specific Rubisco small subunit, NtRbcS-T, which belongs to an uncharacterized phylogenetic cluster (T). This cluster contains RbcS from at least 33 species, including monocots, many of which are known to possess glandular trichomes. Cluster T is distinct from the cluster M which includes the abundant, functionally characterized RbcS isoforms expressed in mesophyll or bundle-sheath cells. Expression of NtRbcS-T in Chlamydomonas and purification of the full Rubisco complex showed that this isoform conferred higher Vmax and Km values as well as higher acidic pH-dependent activity than NtRbcS-M, an isoform expressed in the mesophyll. This observation was confirmed with trichome extracts. These data show that an ancient divergence allowed for the emergence of a so far uncharacterized RbcS cluster. We propose that secretory trichomes have a particular Rubisco uniquely adapted to secretory cells where CO2 is released by the active specialized metabolism.
Main conclusion:
Production of compound K (a ginsenoside saponin) and its precursors in transgenic tobacco resulted in stunted growth and seed set failure, which may be caused by strong autotoxicity of heterologously produced phytochemicals against the tobacco itself. Panax ginseng roots contain various saponins (ginsenosides), which are major bioactive compounds. A monoglucosylated saponin, compound K (20-O-(β-D-glucopyranosyl)-20(S)-protopanaxadiol), has high medicinal and cosmetic values but is present in undetectable amounts in naturally grown ginseng roots. The production of compound K (CK) requires complicated deglycosylation of ginsenosides using physicochemical and/or enzymatic degradation. In this work, we report the production of CK in transgenic tobacco by co-overexpressing three genes (PgDDS, CYP716A47 and UGT71A28) isolated from P. ginseng. Introduction and expression of the transgenes in tobacco lines were confirmed by genomic PCR and RT-PCR. All the lines of transgenic tobacco produced CK including its precursors, protopanaxadiol and dammarenediol-II (DD). The concentrations of CK in the leaves ranged from 1.55 to 2.64 µg/g dry weight, depending on the transgenic line. Interestingly, production of CK in tobacco brought stunted plant growth and gave rise to seed set failure. This seed set failure was caused by both long-styled flowers and abnormal pollen development in transgenic tobacco. Both CK and DD treatments highly suppressed in vitro germination and tube growth in wild-type pollens. Based on these results, metabolic engineering for CK production in transgenic tobacco was successfully achieved, but the production of CK and its precursors in tobacco severely affects vegetative and reproductive growth due to the cytotoxicity of phytochemicals that are heterologously produced in transgenic tobacco.
Active transport of aromas
Volatile organic compounds (VOCs) serve as invisible lines of communication among host plants, pathogens, commensals, community groups, and, with flowers, their pollinators. Studying petunia flowers, Adebesin et al. show that VOCs do not passively diffuse out of the cells but are actively shuttled across the plasma membrane by an ABC (ATP-binding cassette) transporter (see the Perspective by Eberl and Gershenzon). Disabling the transporter results in damage to the cell's membranes by intracellular accumulation of VOCs.
Science , this issue p. 1386 ; see also p. 1334
Glandular trichomes are metabolic cell factories with the capacity to produce large quantities of secondary metabolites. Little is known about the connection between central carbon metabolism and metabolic productivity for secondary metabolites in glandular trichomes. To address this gap in our knowledge, we performed comparative metabolomics, transcriptomics, proteomics and 13C-labeling of type VI glandular trichomes and leaves from a cultivated (Solanum lycopersicum LA4024) and a wild (Solanum habrochaites LA1777) tomato accession. Specific features of glandular trichomes that drive the formation of secondary metabolites could be identified. Tomato type VI trichomes are photosynthetic but acquire their carbon essentially from leaf sucrose. The energy and reducing power from photosynthesis are used to support the biosynthesis of secondary metabolites, while the comparatively reduced Calvin-Benson-Bassham cycle activity may be involved in recycling metabolic CO2. Glandular trichomes cope with oxidative stress by producing high levels of polyunsaturated fatty acids, oxylipins, and glutathione. Finally, distinct mechanisms are present in glandular trichomes to increase the supply of precursors for the isoprenoid pathways. Particularly, the citrate-malate shuttle supplies cytosolic acetyl CoA and plastidic glycolysis and malic enzyme support the formation of plastidic pyruvate. A model is proposed on how glandular trichomes achieve high metabolic productivity.
O-acyl sugars (O-AS) are abundant trichome-specific metabolites that function as indirect defenses against herbivores of the wild tobacco Nicotiana attenuata; whether they also function as generalized direct defenses against herbivores and pathogens remains unknown. We characterized natural variation in O-AS among 26 accessions and examined their influence on two native fungal pathogens, Fusarium brachygibbosum U4 and Alternaria sp. U10, and the specialist herbivore, Manduca sexta. At least 15 different O-AS structures belonging to three classes were found in N. attenuata leaves. A three-fold quantitative variation in total leaf O-AS was found among the natural accessions. Experiments with natural accessions and crosses between high- and low-O-AS accessions revealed that total O-AS levels were associated with resistance against herbivores and pathogens. Removing O-AS from the leaf surface increased M. sexta growth rate and plant fungal susceptibility. O-AS supplementation in artificial diets and germination medium reduced M. sexta growth and fungal spore germination, respectively. Finally, silencing the expression of a putative branched-chain alpha-ketoacid dehydrogenase E1 beta subunit encoding gene (NaBCKDE1B) in the trichomes reduced total leaf O-AS by 20-30% and increased susceptibility to Fusarium pathogens. We conclude that O-AS function as direct defenses to protect plants from attack from both native pathogenic fungi and a specialist herbivore, and infer that their diversification is likely shaped by the functional interactions among these biotic stresses.
The effective anti-malarial drug artemisinin (AN) isolated from Artemisia annua is relatively expensive due to the low content in the plant as it is only synthesized within the glandular trichomes. Therefore, genetic engineering of A. annua is one of the most promising approaches to improve AN yield. In this work, AaMYB1 transcription factor has been identified and characterized. When AaMYB1 is overexpressed in A. annua, either exclusively in trichomes or in the whole plant, essential AN biosynthetic genes are also overexpressed and consequently AN amount significantly increased. Artemisia AaMYB1 constitutively overexpressing plants displayed a higher number of trichomes. In order to study this role on trichome development and others possibly connected biological processes, AaMYB1 was overexpressed in Arabidopsis thaliana. For supporting our findings in A. thaliana, AaMYB1 orthologue from this model plant AtMYB61 was identified and atmyb61 mutants characterized. Both AaMYB1 and AtMYB61 affected trichome initiation, root development and stomatal aperture in A. thaliana. Molecular analyses indicated that two crucial trichome activator genes are misexpressed in atmyb61 mutant plants and in plants overexpressing AaMYB1. Furthermore, AaMYB1 and AtMYB61 are also essential for gibberellin (GA) biosynthesis and degradation in both species by positively affecting the expression of the enzymes that convert GA9 into the bioactive GA4 as well as the enzymes involved in the degradation of GA4 . Overall, these results identify AaMYB1/AtMYB61 as a key component of the molecular network that connect important biosynthetic processes, and reveal its potential value for AN production through genetic engineering. This article is protected by copyright. All rights reserved.