AaCycTL Regulates Cuticle and Trichome Development in Arabidopsis and Artemisia annua L.

Artemisinin is an important drug for resistance against malaria. Artemisinin is derived from the glandular trichome of leaves, stems, or buds of the Chinese traditional herb Artemisia annua. Increasing the trichome density may enhance the artemisinin content of A. annua. It has been proven that cyclins are involved in the development of trichomes in tomato, Arabidopsis, and tobacco, but it is unclear whether the cyclins in A. annua influence trichome development. In this study, we showed that AaCycTL may regulate trichome development and affect the content of artemisinin. We cloned AaCycTL and found that it has the same expression files as the artemisinin biosynthesis pathway gene. We overexpressed AaCycTL in Arabidopsis, and the results indicated that AaCycTL changed the wax coverage on the surface of Arabidopsis leaves. The trichome density decreased as well. Using yeast two-hybrid and BiFC assays, we show that AaCycTL can interact with AaTAR1. Moreover, we overexpressed AaCycTL in A. annua and found that the expression of AaCycTL was increased to 82–195%. Changes in wax coverage on the surface of transgenic A. annua leaves or stems were found as well. We identified the expression of the artemisinin biosynthesis pathway genes ADS, CYP71AV1, and ALDH1 has decreased to 88–98%, 76–97%, and 82–97% in the AaCycTL-overexpressing A. annua lines, respectively. Furthermore, we found reduced the content of artemisinin. In agreement, overexpression of AaCycTL in A. annua or Arabidopsis may alter waxy loading, change the initiation of trichomes and downregulate trichome density. Altogether, AaCycTL mediates trichome development in A. annua and thus may serve to regulate trichome density and be used for artemisinin biosynthesis.

Hairiness Gene Regulated Multicellular, Non-Glandular Trichome Formation in Pepper Species

Trichomes are unicellular or multicellular epidermal structures that play a defensive role against environmental stresses. Although unicellular trichomes have been extensively studied as a mechanistic model, the genes involved in multicellular trichome formation are not well understood. In this study, we first classified the trichome morphology structures in Capsicum species using 280 diverse peppers. We cloned a key gene (Hairiness) on chromosome 10, which mainly controlled the formation of multicellular non-glandular trichomes (types II, III, and V). Hairiness encodes a Cys2-His2 zinc-finger protein, and virus-induced gene silencing of the gene resulted in a hairless phenotype. Differential expression of Hairiness between the hairiness and hairless lines was due to variations in promoter sequences. Transgenic experiments verified the hypothesis that the promoter of Hairiness in the hairless line had extremely low activity causing a hairless phenotype. Hair controlled the formation of type I glandular trichomes in tomatoes, which was due to nucleotide differences. Taken together, our findings suggest that the regulation of multicellular trichome formation might have similar pathways, but the gene could perform slightly different functions in crops.

Developmental regulation of lupulin gland-associated genes in aromatic and bitter hops (Humulus lupulus L.)

Background Hop (Humulus lupulus L.) bitter acids are valuable metabolites for the brewing industry. They are biosynthesized and accumulate in glandular trichomes of the female inflorescence (hop cone). The content of alpha bitter acids, such as humulones, in hop cones can differentiate aromatic from bitter hop cultivars. These contents are subject to genetic and environmental control but significantly correlate with the number and size of glandular trichomes (lupulin glands). Results We evaluated the expression levels of 37 genes involved in bitter acid biosynthesis and morphological and developmental differentiation of glandular trichomes to identify key regulatory factors involved in bitter acid content differences. For bitter acid biosynthesis genes, upregulation of humulone synthase genes, which are important for the biosynthesis of alpha bitter acids in lupulin glands, could explain the higher accumulation of alpha bitter acids in bitter hops. Several transcription factors, including HlETC1, HlMYB61 and HlMYB5 from the MYB family, as well as HlGLABRA2, HlCYCB2–4, HlZFP8 and HlYABBY1, were also more highly expressed in the bitter hop cultivars; therefore, these factors may be important for the higher density of lupulin glands also seen in the bitter hop cultivars. Conclusions Gene expression analyses enabled us to investigate the differences between aromatic and bitter hops. This study confirmed that the bitter acid content in glandular trichomes (lupulin glands) is dependent on the last step of alpha bitter acid biosynthesis and glandular trichome density.

Delineating the genetic regulation of cannabinoid biosynthesis during female flower development in Cannabis sativa

Cannabinoids are predominantly produced in the glandular trichomes on cannabis female flowers. There is little known on how cannabinoid biosynthesis is regulated during female flower development. We aim to understand the rate-limiting step(s) in the cannabinoid biosynthetic pathway. We investigated the transcript levels of cannabinoid biosynthetic genes as well as cannabinoid contents during 7 weeks of female flower development. We demonstrated that the enzymatic steps for producing CBG, which involve genes GPPS, PT and OAC, could be rate limit cannabinoid biosynthesis. Our findings further suggest that cannabinoid synthases, CBDAS and THCAS in a hemp and medical marijuana variety respectively, are not critical for cannabinoid biosynthesis. The cannabinoid biosynthetic genes are generally upregulated during flower maturation, which indicate glandular trichome development. MeJA can potentially increase cannabinoid production. We propose that biweekly application of 100 μM MeJA staring from flower initiation would be efficacious for promoting cannabinoid biosynthesis.

PHARMACOGNOSTICAL AND PHYTOCHEMICAL STUDY ON LEAF OF COMBRETUM INDICUM (L.)

Combretum indicum (L.) is an extra-pharmacopeial drug with an abundance of medicinal properties. Throughout the world, different Parts of Combretum indicum (L.) is used in curing many ailments by folklore healers. In India, the drug Combretum indicum (L.) is commonly known by the name MadhuMalathi and used by the folklore healers of different areas. Especially the use of Combretum indicum (L.) is seen among the folklore Practitioners of Dakshina Kannada District in curing ailments including paediatric conditions. Thus, it is very important to explore the identity of this drug by the proper Pharmacognostical and Phytochemical analysis. Hence, the present study was aimed at Pharmacognostical and Phytochemical study on Leaf of Combretum indicum (L.). The transverse section of the midrib of the leaf shows the presence of the endodermal layer; which is single-layered, surrounds by a vascular bundle and packed with starch grains. Endodermis covers the vascular bundle and contains several starch grains. The powder microscopy shows the presence of glandular trichome, calcium oxalate crystals etc. The percentage of extractive value was maximum in methanol which was 7.18%, Preliminary phytochemical study showed the presence of Proteins, Carbohydrates, Tannins, Steroids, Alkaloids, Triterpenoids, Starch, Resin and Phenols. The total ash attained was 7.84% and the water-soluble ash was 3.4%.

Hair (H) interacts with SlZFP8-like to regulate the initiation and elongation of trichomes by modulating SlZFP6 expression in tomato

Trichomes are specialized glandular or non-glandular structures that provide physical or chemical protection against insect and pathogens attack. Trichomes in Arabidopsis, as typical non-glandular structures, have been extensively studied. However, the molecular mechanism underlying glandular trichome formation and elongation still remains largely unknown. We previously demonstrated that Hair (H) is essential for the formation of type I and type VI trichomes. Here, we found that overexpression of H increased the density and length of tomato trichomes. We revealed that H physically interacts with its close homolog SlZFP8-like (SlZFP8L) and SlZFP8L also directly interacts with Woolly (Wo) by biochemical assays. SlZFP8L overexpression plants showed increased trichome density and length. We further found that the expression of SlZFP6, encoding a C2H2 zinc finger protein, is positively regulated by H. We identified that SlZFP6, is a direct target of H through ChIP-qPCR, Y1H, and LUC assays. Similar to H and SlZFP8L, the overexpression of SlZFP6 also increased the density and length of tomato trichomes. Taken together, our results suggest that H interacts with SlZFP8-like to regulate the initiation and elongation of trichomes by modulating SlZFP6 expression in tomato.

Microscopical Characterization and Physicochemical Standardization of Leaves, Stems and Roots of Spondias mombin L. (Anacardiaceae)

Background: Spondias mombin L. belongs to the family Anacardiaceae. Despite its wide ethnomedicinal applications in the management of diverse diseases, there is a paucity of documented reports on its standardization.Objectives: The present study evaluated microscopical characters and some physicochemical properties of different parts of the plant for its identification and standardization.Material and Methods: Epidermal tissue preparation of the leaf of Spondias mombin (SM) was obtained using physical method while thin sections (10-12 μm) of the stem bark and root were obtained using a rotary microtome. Physicochemical parameters were determined for the powdered samples of SM using standard methods.Results: Diagnostic characters from the epidermal tissue of the leaf revealed anomocytic, paracytic stomatal type, non-glandular trichome, smooth to slightly wavy anticlinal walls while sections of the stem bark and root were characterized with abundant sclereids and calcium oxalate crystals. The stomatal number and stomatal index of the abaxial epidermis were 23.70±0.86 and 24.62±0.78 %, respectively. Ethanol had the highest extractive value (17.84±0.50) % in the leaf whereas it was lowest in petroleum ether (1.92±0.08) %. The leaf had the lowest ash value (7.13±0.76) %.Conclusion: The microscopical characterization and some of the physicochemical parameters reported herein could be useful in the compilation of monograph for the correct identification of Spondias mombin, thus contributing to the knowledge of its collection and preservation.

Single Wavelengths of LED Light Supplement Promote the Biosynthesis of Major Cyclic Monoterpenes in Japanese Mint

Environmental light conditions influence the biosynthesis of monoterpenes in the mint plant. Cyclic terpenes, such as menthol, menthone, pulegone, and menthofuran, are major odor components synthesized in mint leaves. However, it is unclear how light for cultivation affects the contents of these compounds. Artificial lighting using light-emitting diodes (LEDs) for plant cultivation has the advantage of preferential wavelength control. Here, we monitored monoterpene contents in hydroponically cultivated Japanese mint leaves under blue, red, or far-red wavelengths of LED light supplements. Volatile cyclic monoterpenes, pulegone, menthone, menthol, and menthofuran were quantified using the head-space solid phase microextraction method. As a result, all light wavelengths promoted the biosynthesis of the compounds. Remarkably, two weeks of blue-light supplement increased all compounds: pulegone (362% increase compared to the control), menthofuran (285%), menthone (223%), and menthol (389%). Red light slightly promoted pulegone (256%), menthofuran (178%), and menthol (197%). Interestingly, the accumulation of menthone (229%) or menthofuran (339%) was observed with far-red light treatment. The quantification of glandular trichomes density revealed that no increase under light supplement was confirmed. Blue light treatment even suppressed the glandular trichome formation. No promotion of photosynthesis was observed by pulse-amplitude-modulation (PAM) fluorometry. The present result indicates that light supplements directly promoted the biosynthetic pathways of cyclic monoterpenes.

Micromorphological variations of trichomes in the genus Ocimum L.

Ocimum L. is an attractive fragrant ornamental plant with medicinal aromatic compounds. The study using digital microscopy revealed the distinct epidermal trichome morphology in five species of Ocimum including O. americanum L., O. basilicum L., O. gratissimum L., O. kilimandscharicum Gurke and O. tenuiflorum L. A distinguished variation in size, shape, type and abundance of trichomes on leaves and stem of all species are described and photographed. Both glandular and non-glandular trichomes scrutinized in all the species in which glandular type comprised capitate and peltate trichomes. On the stem, the longest glandular capitate trichome (GCT) and non-glandular trichome (NGT) were observed in O. tenuiflorum while O. basilicum possessed longest glandular peltate trichome (GPT). In case of foliar trichomes, the longest GCT observed on leaves of O. americanum; and O. basilicum showed the longest GPT and NGT. In the species, the septate or aseptate NGT possessed uni, bi or multicellular base with acute or broad apex. A distinguished variation in the number of cells in stalk and head of GT was observed in the study. Presence of silica bodies in peltate trichome of O. kilimandscharicum was noticed as the characteristic feature. Among the five species, the trichomes were abundant in O. kilimandscharicum; while O. basilicum showed sparse distribution. Trichomes with disrupted cells were also noted in O. americanum. Due to the presence of specific trichome characteristics, the efficient taxonomic key is prepared to identify the taxa at species level.