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Exploration of alternative sources of antibacterial compounds is an important and possibly an effective solution to the current challenges in antimicrobial therapy. Plant derived wastes may offer one such alternative. Here, we investigated the antibacterial property of extract derived from a part of the Jackfruit (Artocarpus heterophyllus Lam.) called 'rag', generally considered as fruit waste. Morpho-physical characterization of the Jackfruit rag extract (JFRE) was performed using Gaschromatography, where peaks indicative of furfural; pentanoic acid; and hexadecanoic acid were observed. In vitro biocompatibility of JFRE was performed using the MTT assay, which showed comparable cellular viability between extracttreated and untreated mouse fibroblast cells. Agar well disc diffusion assay exhibited JFRE induced zones of inhibition for a wide variety of laboratory and clinical strains of Gram-positive and Gram-negative bacteria. Analysis of electron microscope images of bacterial cells suggests that JFRE induces cell death by disintegration of the bacterial cell wall and precipitating intracytoplasmic clumping. The antibacterial activity of the JFREs was further validated in vivo using Shigella dysenteriae infected fly model, where JFRE pre-fed flies infected with S. dysenteriae had significantly reduced mortality compared to controls. JFRE demonstrates broad antibacterial property, both in vitro and in vivo, possibly by its activity on bacterial cell wall. [ABSTRACT FROM AUTHOR]

Terpene synthases (TPSs) produce a variety of terpenoids that play numerous functional roles in primary and secondary metabolism, as well as in ecological interactions. Here, we report the functional characterization of an inducible potato TPS gene encoding bulnesol/elemol synthase (StBUS/ELS). The expression of StBUS/ELS in potato leaves was significantly induced in response to both bacterial (Pseudomonas syringae) and fungal (Alternaria solani) infection as well as methyl jasmonate treatment, indicating its role in defense. The leaves exhibited the highest StBUS/ELS expression followed by the stem with least and similar expression in tuber, sprout and root. Recombinant StBUS/ELS catalyzed the formation of different sesquiterpenes by utilizing farnesyl diphosphate as substrate, and the monoterpene geraniol from geranyl diphosphate. Among the sesquiterpenes formed by StBUS/ELS, elemol was the predominant product followed by α‐bulnesene, bulnesol and β‐elemene. Further gas chromatography–mass spectrometry (GC–MS) analysis of StBUS/ELS assay products at different injection temperatures revealed elemol and bulnesol as the major products at 275 and 200/150°C, respectively, without much change in the levels of minor products. This indicated thermal rearrangement of bulnesol into elemol at higher temperatures. Transient overexpression of StBUS/ELS in potato leaves conferred tolerance against the growth of bacteria P. syringae and Ralstonia solanacearum, and the fungus A. solani. Further, expression analysis of pathogenesis‐related (PR) genes in StBUS/ELS overexpressing leaves showed no significant change in comparison to control, indicating a direct involvement of StBUS/ELS enzymatic products against pathogens. Overall, our study suggested that StBUS/ELS is a pathogen‐inducible TPS encoding bulnesol/elemol synthase and could provide a direct role in defense against biotic stress in potato. [ABSTRACT FROM AUTHOR]

The medicinal properties of Ashwagandha (Withania somnifera) are accredited to a group of compounds called withanolides. 24-Methylene cholesterol is the intermediate for sterol biosynthesis and a proposed precursor of withanolide biogenesis. However, conversion of 24-methylene cholesterol to withaferin A and other withanolides has not yet been biochemically dissected. Hence, in an effort to fill this gap, an important gene, encoding S -adenosyl l -methionine-dependent sterol-C24-methyltransferase type 1 (SMT1), involved in the first committed step of sterol biosynthesis, from W. somnifera was targeted in the present study. Though SMT1 has been characterized in model plants such as Nicotiana tabacum and Arabidopsis thaliana, its functional role in phytosterol and withanolide biosynthesis was demonstrated for the first time in W. somnifera. Since SMT1 acts at many steps preceding the withanolide precursor, the impact of this gene in channeling of metabolites for withanolide biosynthesis and its regulatory nature was illustrated by suppressing the gene in W. somnifera via the RNA interference (RNAi) approach. Interestingly, down-regulation of SMT1 in W. somnifera led to reduced levels of campesterol, sitosterol and stigmasterol, with an increase of cholesterol content in the transgenic RNAi lines. In contrast, SMT1 overexpression in transgenic N. tabacum enhanced the level of all phytosterols except cholesterol, which was not affected. The results established that SMT1 plays a crucial role in W. somnifera withanolide biosynthesis predominantly through the campesterol and stigmasterol routes. [ABSTRACT FROM AUTHOR]

Catharanthus roseus is the sole source of two of the most important anticancer monoterpene indole alkaloids (MIAs), vinblastine and vincristine and their precursors, vindoline and catharanthine. The MIAs are produced from the condensation of precursors derived from indole and terpene secoiridoid pathways. It has been previously reported that the terpene moiety limits MIA biosynthesis in C. roseus. Here, to overcome this limitation and enhance MIAs levels in C. roseus, bifunctional geranyl(geranyl) diphosphate synthase [G(G)PPS] and geraniol synthase (GES) that provide precursors for early steps of terpene moiety (secologanin) formation, were overexpressed transiently by agroinfiltration and stably by Agrobacterium-mediated transformation. Both transient and stable overexpression of G(G)PPS and co-expression of G(G)PPS+GES significantly enhanced the accumulation of secologanin, which in turn elevated the levels of monomeric MIAs. In addition, transgenic C. roseus plants exhibited increased levels of root alkaloid ajmalicine. The dimeric alkaloid vinblastine was enhanced only in G(G)PPS but not in G(G)PPS+GES transgenic lines that correlated with transcript levels of peroxidase-1 (PRX1) involved in coupling of vindoline and catharanthine into 3′,4′-anhydrovinblastine, the immediate precursor of vinblastine. Moreover, first generation (T1) lines exhibited comparable transcript and metabolite levels to that of T0 lines. In addition, transgenic lines displayed normal growth similar to wild-type plants indicating that the bifunctional G(G)PPS enhanced flux toward both primary and secondary metabolism. These results revealed that improved availability of early precursors for terpene moiety biosynthesis enhanced production of MIAs in C. roseus at the whole plant level. This is the first report demonstrating enhanced accumulation of monomeric and dimeric MIAs including root MIA ajmalicine in C. roseus through transgenic approaches. [ABSTRACT FROM AUTHOR]

Withania somnifera produces pharmacologically important triterpenoid withanolides that are derived via phytosterol pathway; however, their biosynthesis and regulation remain to be elucidated., A jasmonate- and salicin-inducible WRKY transcription factor from W. somnifera ( Ws WRKY1) exhibiting correlation with withaferin A accumulation was functionally characterized employing virus-induced gene silencing and overexpression studies combined with transcript and metabolite analyses, and chromatin immunoprecipitation assay., Ws WRKY1 silencing resulted in stunted plant growth, reduced transcripts of phytosterol pathway genes with corresponding reduction in phytosterols and withanolides in W. somnifera. Its overexpression elevated the biosynthesis of triterpenoids in W. somnifera (phytosterols and withanolides), as well as tobacco and tomato (phytosterols). Moreover, Ws WRKY1 binds to W-box sequences in promoters of W. somnifera genes encoding squalene synthase and squalene epoxidase, indicating its direct regulation of triterpenoid pathway. Furthermore, while Ws WRKY1 silencing in W. somnifera compromised the tolerance to bacterial growth, fungal infection, and insect feeding, its overexpression in tobacco led to improved biotic stress tolerance., Together these findings demonstrate that Ws WRKY1 has a positive regulatory role on phytosterol and withanolides biosynthesis, and defense against biotic stress, highlighting its importance as a metabolic engineering tool for simultaneous improvement of triterpenoid biosynthesis and plant defense. [ABSTRACT FROM AUTHOR]

Summary Brassica juncea 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) is encoded by four isogenes ( BjHMGS1- BjHMGS4). In vitro enzyme assays had indicated that the recombinant BjHMGS1 H188N mutant lacked substrate inhibition by acetoacetyl-CoA (AcAc-CoA) and showed 8-fold decreased enzyme activity. The S359A mutant demonstrated 10-fold higher activity, while the H188N/S359A double mutant displayed a 10-fold increased enzyme activity and lacked inhibition by AcAc-CoA. Here, wild-type and mutant BjHMGS1 were overexpressed in Arabidopsis to examine their effects in planta. The expression of selected genes in isoprenoid biosynthesis, isoprenoid content, seed germination and stress tolerance was analysed in HMGS overexpressors (OEs). Those mRNAs encoding enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), sterol methyltransferase 2 (SMT2), delta-24 sterol reductase (DWF1), C-22 sterol desaturase (CYP710A1) and brassinosteroid-6-oxidase 2 (BR6OX2) were up-regulated in HMGS-OEs. The total sterol content in leaves and seedlings of OE-wtBjHMGS1, OE-S359A and OE-H188N/S359A was significantly higher than OE-H188N. HMGS-OE seeds germinated earlier than wild-type and vector-transformed controls. HMGS-OEs further displayed reduced hydrogen peroxide (H2O2)-induced cell death and constitutive expression of salicylic acid (SA)-dependent pathogenesis-related genes ( PR1, PR2 and PR5), resulting in an increased resistance to Botrytis cinerea, with OE-S359A showing the highest and OE-H188N the lowest tolerance. These results suggest that overexpression of HMGS up-regulates HMGR, SMT2, DWF1, CYP710A1 and BR6OX2, leading to enhanced sterol content and stress tolerance in Arabidopsis. [ABSTRACT FROM AUTHOR]

Geranyl diphosphate (GPP), the precursor of many monoterpene end products, is synthesized in plastids by a condensation of dimethylallyl diphosphate and isopentenyl diphosphate (IPP) in a reaction catalyzed by homodimeric or heterodimeric GPP synthase (GPPS). In the heterodimeric enzymes, a noncatalytic small subunit (GPPS.SSU) determines the product specificity of the catalytic large subunit, which may be either an active geranylgeranyl diphosphate synthase (GGPPS) or an inactive GGPPS-like protein. Here, we show that expression of snapdragon (Antirrhinum majus) GPPS.SSU in tobacco (Nicotiana tabacum) plants increased the total GPPS activity and monoterpene emission from leaves and flowers, indicating that the introduced catalytically inactive GPPS.SSU found endogenous large subunit partner(s) and formed an active snapdragon/tobacco GPPS in planta. Bimolecular fluorescence complementation and in vitro enzyme analysis of individual and hybrid proteins revealed that two of four GGPPS-like candidates from tobacco EST databases encode bona fide GGPPS that can interact with snapdragon GPPS.SSU and form a functional GPPS enzyme in plastids. The formation of chimeric GPPS in transgenic plants also resulted in leaf chlorosis, increased light sensitivity, and dwarfism due to decreased levels of chlorophylls, carotenoids, and gibberellins. In addition, these transgenic plants had reduced levels of sesquiterpene emission, suggesting that the export of isoprenoid intermediates from the plastids into the cytosol was decreased. These results provide genetic evidence that GPPS.SSU modifies the chain length specificity of phylogenetically distant GGPPS and can modulate IPP flux distribution between GPP and GGPP synthesis in planta. [ABSTRACT FROM AUTHOR]

Brassica juncea BjCHI1 is a plant chitinase with two chitin-binding domains. Its expression, induced in response to wounding, methyl jasmonate treatment, Aspergillus niger infection, and caterpillar Pieris rapae feeding, suggests that it plays a role in defence. In this study, to investigate the potential of using BjCHI1 in agriculture, Pichia-expressed BjCHI1 and its deletion derivatives that lack one or both chitin-binding domains were tested against phytopathogenic fungi and bacteria. Transplastomic tobacco expressing BjCHI1 was also generated and its extracts assessed. In radial growth-inhibition assays, BjCHI1 and its derivative with one chitin-binding domain showed anti-fungal activities against phytopathogens, Colletotrichum truncatum, C. acutatum, Botrytis cinerea, and Ascochyta rabiei. BjCHI1 also inhibited spore germination of C. truncatum. Furthermore, BjCHI1, but not its derivatives lacking one or both domains, inhibited the growth of Gram-negative bacteria (Escherichia coli, Ralstonia solanacearum, Pseudomonas aeruginosa) more effectively than Gram-positive bacteria (Micrococcus luteus and Bacillus megaterium), indicating that the duplicated chitin-binding domain, uncommon in chitinases, is essential for bacterial agglutination. Galactose, glucose, and lactose relieved agglutination, suggesting that BjCHI1 interacts with the carbohydrate components of the Gram-negative bacterial cell wall. Retention of chitinase and bacterial agglutination activities in transplastomic tobacco extracts implicates that BjCHI1 is potentially useful against both fungal and bacterial phytopathogens in agriculture. [ABSTRACT FROM PUBLISHER]

Ashwagandha (Withania somnifera L. Dunal) is a versatile medicinal plant of Solanaceae family, renowned for its potent therapeutic properties, due to which it is extensively used in Indian traditional systems of medicine such as Ayurveda. The medicinal properties are attributed to specialized metabolites known as withanolides, which are chemically triterpenoid steroidal lactones. Despite their significance, the biosynthetic pathway of withanolides remains poorly understood. It is hypothesized that withanolides are synthesized through the universal sterol pathway, wherein sterol precursors undergo various biochemical modifications such as hydroxylation, oxidation, cyclization, and glycosylation, yielding a diverse array of downstream withanolides and withanosides. Consequently, comprehending the biosynthetic pathway of withanolides is crucial to facilitate advancements in withanolides productivity through metabolic engineering or synthetic biology approaches. This article aims to provide an update on the efforts made toward understanding withanolides formation and regulation and highlights gaps and approaches to elucidate the withanolides biosynthesis in W. somnifera., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Virus-induced gene silencing (VIGS) is a functional genomics tool to transiently downregulate the expression of target gene(s) by exploiting the plant's innate defense mechanism against invading RNA viruses. VIGS is a rapid and efficient approach to analyze the gene function, particularly, in the plants that are not amenable to stable genetic transformation. This strategy has been successfully used to decipher the function of several genes and transcription factors involved in the biosynthesis of specialized metabolites and regulation of specialized metabolism, respectively, in different medicinal and aromatic plants. Here, we describe a detailed Tobacco rattle virus (TRV)-mediated VIGS protocol for silencing of the gene encoding Phytoene desaturase (PDS) in important medicinal plants Catharanthus roseus, Calotropis gigantean, Rauwolfia serpentina, and Ocimum basilicum. Our methods allow the study of gene function within 3-4 weeks after agro-inoculation, and can be an easy and efficient approach for future studies on understanding of the biosynthesis of specialized metabolites in these important medicinal plants., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Madagascar periwinkle (Catharanthus roseus, family Apocynaceae) is a reservoir of more than 130 monoterpene indole alkaloids (MIAs) including the famous anti-neoplastic dimeric MIAs vinblastine and vincristine, and anti-hypertensive monomeric MIAs ajmalicine and serpentine. Understanding the biosynthetic steps and regulatory factors leading to the formation of MIAs is crucial for rational engineering to achieve targeted enhancement of different MIAs. Due to its highly recalcitrant nature, C. roseus is considered genetically non-tractable for transformation at the whole-plant level. Though few reports have demonstrated tissue culture-mediated regeneration and transformation of C. roseus at whole-plant level recently, the efficiency and reproducibility of these protocols have been a major challenge. To overcome this, we have developed a tissue-culture-independent Agrobacterium-mediated in planta transformation method in C. roseus. Using this method, we were able to efficiently generate stable transgenic plants without relying on the cumbersome methods of tissue-culture regeneration and transformation. Moreover, the transformed plants obtained through this in planta method exhibited stability in subsequent generations. Our method is useful not only for the elucidation of biosynthetic and regulatory steps involved in MIA formation through transgenic plant approach but also for metabolic engineering at the whole-plant level in C. roseus., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Deglycosylation is a key step in the activation of specialized metabolites involved in plant defense mechanisms. This reaction is notably catalyzed by β-glucosidases of the glycosyl hydrolase 1 (GH1) family such as strictosidine β-d-glucosidase (SGD) from Catharanthus roseus. SGD catalyzes the deglycosylation of strictosidine, forming a highly reactive aglycone involved in the synthesis of cytotoxic monoterpene indole alkaloids (MIAs) and in the crosslinking of aggressor proteins. By exploring C. roseus transcriptomic resources, we identified an alternative splicing event of the SGD gene leading to the formation of a shorter isoform of this enzyme (shSGD) that lacks the last 71-residues and whose transcript ratio with SGD ranges from 1.7% up to 42.8%, depending on organs and conditions. Whereas it completely lacks β-glucosidase activity, shSGD interacts with SGD and causes the disruption of SGD multimers. Such disorganization drastically inhibits SGD activity and impacts downstream MIA synthesis. In addition, shSGD disrupts the metabolic channeling of downstream biosynthetic steps by hampering the recruitment of tetrahydroalstonine synthase in cell nuclei. shSGD thus corresponds to a pseudo-enzyme acting as a regulator of MIA biosynthesis. These data shed light on a peculiar control mechanism of β-glucosidase multimerization, an organization common to many defensive GH1 members., (© American Society of Plant Biologists 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Virus-induced gene silencing (VIGS) has emerged as a fast and efficient reverse and forward genetics tool to study gene function in model plants as well as in agriculturally important plants. In addition, VIGS approach has been successfully used to provide insights into the role of several genes and regulators involved in plant secondary metabolism. Ashwagandha (Withania somnifera) is an important Indian medicinal plant that accumulates pharmacologically important triterpenoid steroidal lactones, which are collectively termed as withanolides. W. somnifera being a highly recalcitrant plant for genetic transformation, Tobacco rattle virus (TRV)-mediated VIGS was established by our group to facilitate understanding of withanolides' pathway. Here, we describe a detailed procedure to carry out VIGS for gene function studies in W. somnifera.