High-value Secondary Metabolites Research Articles

Comprehensive genome analysis of two novel Saccharopolyspora strains-Saccharopolyspora montiporae sp. nov. and Saccharopolyspora galaxeae sp. nov. isolated from stony corals in Hainan.

Marine actinomycetes exhibit a high level of biodiversity and possess significant potential for the production of high-value secondary metabolites. During the course of investigation of marine actinobacteria from corals, two Saccharopolyspora strains, namely, HNM0983T and HNM0986T, were isolated from stony corals collected from the coastal area of Hainan Island. The 16S ribosomal RNA (rRNA) gene sequence analysis revealed that these two strains are putative novel taxa of the genus Saccharopolyspora. Whole-genome sequencing comparisons further confirmed the two strains as belonging to two novel Saccharopolyspora species, which can be distinguished phenotypically and chemically from their current closest phylogenetic relatives. Some genomic information of the genus Saccharopolyspora was compared for evaluating the production capacity of secondary metabolites. A total of 519 biosynthetic gene clusters (BGCs) from the genus Saccharopolyspora were used for analysis, and terpene BGCs were found to be widespread and most abundant in this genus. In addition, abundant novel BGCs in the genus Saccharopolyspora are not clustered with the known BGCs in the database, indicating that the metabolites of the genus Saccharopolyspora deserve further exploration. On the basis of these presented results, Saccharopolyspora montiporae sp. nov. (type strain = HNM0983T = CCTCC AA 2020014T = KCTC 49526T) and Saccharopolyspora galaxeae sp. nov. (type strain = HNM0986T = CCTCC AA 2020011T = KCTC 49524T) are proposed as the names for the new strains, respectively.

Recent advances in understanding the regulation of plant secondary metabolite biosynthesis by ethylene-mediated pathways.

Plants produce a large repertoire of secondary metabolites. The pathways that lead to the biosynthesis of these metabolites are majorly conserved in the plant kingdom. However, a significant portion of these metabolites are specific to certain groups or species due to variations in the downstream pathways and evolution of the enzymes. These metabolites show spatiotemporal variation in their accumulation and are of great importance to plants due to their role in development, stress response and survival. A large number of these metabolites are in huge industrial demand due to their potential use as therapeutics, aromatics and more. Ethylene, as a plant hormone is long known, and its biosynthetic process, signaling mechanism and effects on development and response pathways have been characterized in many plants. Through exogenous treatments, ethylene and its inhibitors have been used to manipulate the production of various secondary metabolites. However, the research done on a limited number of plants in the last few years has only started to uncover the mechanisms through which ethylene regulates the accumulation of these metabolites. Often in association with other hormones, ethylene participates in fine-tuning the biosynthesis of the secondary metabolites, and brings specificity in the regulation depending on the plant, organ, tissue type and the prevailing conditions. This review summarizes the related studies, interprets the outcomes, and identifies the gaps that will help to breed better varieties of the related crops and produce high-value secondary metabolites for human benefits.

Construction and characterization of a promoter library with varying strengths to enhance acetoin production from xylose in Serratia marcescens.

Serratia marcescens is utilized as a significant enterobacteria in the production of various high-value secondary metabolites. Acetoin serves as a crucial foundational compound of development and finds application in a broad range of fields. Furthermore, S. marcescens HBQA-7 is capable of utilizing xylose as its exclusive carbon source for acetoin production. The objective of this study was to utilize a constitutive promoter screening strategy to enhance both xylose utilization and acetoin production in S. marcescens HBQA-7. By utilizing RNA-seq, we identified the endogenous constitutive promoter P6 that is the most robust, which facilitated the overexpression of the sugar transporter protein GlfL445I , α-acetyl lactate synthase, and α-acetyl lactate decarboxylase, respectively. The resultant recombinant strains exhibited enhanced xylose utilization rates and acetoin yields. Subsequently, a recombinant plasmid, denoted as pBBR1MCS-P6-glfL445I alsSalsD, was constructed, simultaneously expressing the aforementioned three genes. The resulting recombinant strain, designated as S3, demonstrated a 1.89-fold boost in xylose consumption rate compared with the original strain during shake flask fermentation. resulting in the accumulation of 7.14g/L acetoin in the final fermentation medium. Subsequently, in a 5 L fermenter setup, the acetoin yield reached 48.75g/L, corresponding to a xylose-to-acetoin conversion yield of 0.375g/g.

Synthesis and Characterization of a Biopolymer Pectin/Ethanolic Extract from Olive Mill Wastewater: In Vitro Safety and Efficacy Tests on Skin Wound Healing.

Wound-healing delay is one of the major problems of type 2 diabetes, representing also a clinical emergency in non-healing chronic wounds. Natural antioxidants show interesting wound-healing properties, including those extracted from waste derived from olive oil production. Olive mill wastewater is one of the main by-products of the olive oil-making process, and it is rich in high-value secondary metabolites, mainly hydroxytyrosol. We proposed an eco-friendly extraction method, employing both ultrasound-assisted and Soxhlet techniques and ethanol as a solvent, to recover valuable molecules from Roggianella cv (Olea europea L.) olive mill wastewater, which was further entrapped in a pectin polymer via an enzymatic reaction using porcine pancreatic lipase. Pectin, in combination with other substances, promoted and accelerated wound healing and demonstrated good potential to produce a biomedical conjugate for wound treatment. The antioxidant activity of the extracts and conjugate were evaluated against lipophilic (IC50 equal to 0.152 mg mL-1) and hydrophilic (IC50 equal to 0.0371 mg mL-1) radical species as well as the in vitro cytotoxicity via NRU, h-CLAT, and a wound-healing scratch assay and assessment. The pectin conjugate did not exert hemolytic effects on the peripheral blood, demonstrating interesting wound-healing properties due to its ability to stimulate cell proliferation in a dose-dependent manner.

A Comprehensive Review of the Key Characteristics of the Genus Mentha, Natural Compounds and Biotechnological Approaches for the Production of Secondary Metabolites.

The genus Mentha is one of the most aromatic and well-known members of the Lamiaceae family. A wide range of bioactive compounds has been reported in mints. Regarding the high economic importance of Mentha plants due to the presence of valuable metabolites, the demand for their products is growing exponentially. Therefore, to supply such demand, new strategies should be adopted to improve the yield and medicinal quality of the products. The current review is written based on scientific literature obtained from online databases, including Google Scholar, PubMed, Scopus, and Web of Science regarding the characteristic features of some species of the genus Mentha, their distribution and cultivation, main uses and benefits, phytochemical composition, biotechnological approaches for the production of secondary metabolites, and strategies for enhanced production of mints secondary metabolites. In this article, we offer an overview of the key characteristics, natural compounds, biological properties, and medicinal uses of the genus Mentha. Current research describes biotechnological techniques such as in vitro culture methods for the production of high-value secondary metabolites. This review also highlights the strategies such as elicitation, genetic, and metabolic engineering to improve the secondary compounds production level in mint plants. Overall, it can be concluded that identifying the biosynthetic pathways, leading to the accumulation of pharmaceutically important bioactive compounds, has paved the way for developing highly productive mint plants with improved phytochemical profiles.

Suspension culture of somatic embryos for the production of high-value secondary metabolites.

Suspension culture of somatic embryos for the production of high-value secondary metabolites.

From Nature to Lab: A Review of Secondary Metabolite Biosynthetic Pathways, Environmental Influences, and In Vitro Approaches.

Secondary metabolites are gaining an increasing importance in various industries, such as pharmaceuticals, dyes, and food, as is the need for reliable and efficient methods of procuring these compounds. To develop sustainable and cost-effective approaches, a comprehensive understanding of the biosynthetic pathways and the factors influencing secondary metabolite production is essential. These compounds are a unique type of natural product which recognizes the oxidative damage caused by stresses, thereby activating the defence mechanism in plants. Various methods have been developed to enhance the production of secondary metabolites in plants. The elicitor-induced in vitro culture technique is considered an efficient tool for studying and improving the production of secondary metabolites in plants. In the present review, we have documented various biosynthetic pathways and the role of secondary metabolites under diverse environmental stresses. Furthermore, a practical strategy for obtaining consistent and abundant secondary metabolite production via various elicitation agents used in culturing techniques is also mentioned. By elucidating the intricate interplay of regulatory factors, this review paves the way for future advancements in sustainable and efficient production methods for high-value secondary metabolites.

Root predominant overexpression of iaaM and CKX genes promotes root initiation and biomass production in citrus

Promoting initiation and biomass production of roots is significant for plant-based industries including using roots as bioreactors. Two citrus genotypes, Carrizo and US-897, were used as model plants to test the effects of root-predominantly overexpressed the iaaM (indoleacetic acid-tryptophan monooxygenase) gene and a CKX (a cytokinin oxidase/dehydrogenase) gene. The iaaM transgenic lines exhibited markedly faster root initiation, more root numbers, and higher root biomass compared to their wild-type counterparts. The transgenic iaaM + CKX plants also exhibited similar phenotypes, albeit to a lesser extent than the iaaM plants. Molecular analysis revealed an auxin-responsive CsGH3.1 gene was up-regulated in the iaaM roots and iaaM + CKX roots, and a cytokinin-responsive gene CsARR5 gene was down-regulated in the iaaM + CKX roots. Our results demonstrate that root predominant overexpression of the iaaM or both the iaaM and CKX genes drastically enhances the initiation, growth and biomass production of roots. These results provide additional support that manipulation of auxin and cytokinin levels in roots via transgenic or gene-editing technologies may benefit production of high-value secondary metabolites using roots as bioreactors and also improve rooting of recalcitrant plant species.

Plant cell culture technologies: A promising alternatives to produce high-value secondary metabolites

Plants have been used for its medicinal values since ancient time. The medicinal properties of plants are based on their phytochemical constituent particularly secondary metabolites which are produced in low amounts by plants. Secondary metabolites have been used as medicines, flavors, colors, and fragrances. In recent time, these natural compounds are gaining enormous attention in pharmaceutical, cosmetics, and nutraceutical industries and are regarded economically valuable products. The production of plant secondary metabolites in plant is largely dependent on the plant species, environmental factors and geographical regions. In addition, the main challenges in their mass production is reported to be the quality and quantity issues during their synthesis. Therefore, enthusiasm has grown for increasing the production of secondary metabolites by employing in vitro plant cell culture technology and bioengineering methods. Such technological advancement, has led to production of a huge number of medicinal herbs and high-value secondary metabolites that are mostly used in pharmaceuticals, cosmetics and nutraceuticals industries. The current mini-review article focuses on applications of plant cell culture system for the production secondary metabolites and recent techniques used to improve metabolite contents. Furthermore, our review emphasizes safety issues of plant cell culture derived products.

Electromagnetic Field Improved Nanoparticle Impact on Antioxidant Activity and Secondary Metabolite Production in Anthemis gilanica Seedlings

Electromagnetic field (EMF) causes fundamental alternations in biological systems. In this study, we studied the effects of EMF on physiological responses and secondary metabolites production in SiO2 NP-treated Anthemis gilanica plants. The results indicated that EMF improved plant growth by inducing chlorophyll and carotenoid content, which led to enhanced biomass in SiO2 NP-treated plants. EMF enhanced adventitious roots in SiO2 NP-treated plants. EMF treatment improved the activity of antioxidative enzymes such as superoxide dismutase, catalase, and peroxidase in both control and SiO2 NP-treated plants. EMF and SiO2 NP treatments significantly declined hydrogen peroxide content in A. gilanica plants. Although protein content was reduced by SiO2 NP treatment, combined application of EMF with SiO2 NP caused a significant induction in protein content. Our results presented that EMF induced secondary metabolites accumulation such as flavonoid and phenol in SiO2 NP-treated A. gilanica plants. This work can open prospects for the production of the pharmaceutically high-value secondary metabolites.

Identification and selection of reference genes for gene expression analysis by quantitative real-time PCR in Suaeda glauca\u2019s response to salinity

Quantitative real-time polymerase chain reaction (qPCR) using a stable reference gene is widely used for gene expression research. Suaeda glauca L. is a succulent halophyte and medicinal plant that is extensively used for phytoremediation and extraction of medicinal compounds. It thrives under high-salt conditions, which promote the accumulation of high-value secondary metabolites. However, a suitable reference gene has not been identified for gene expression standardization in S. glauca under saline conditions. Here, 10 candidate reference genes, ACT7, ACT11, CCD1, TUA5, UPL1, PP2A, DREB1D, V-H+-ATPase, MPK6, and PHT4;5, were selected from S. glauca transcriptome data. Five statistical algorithms (ΔCq, geNorm, NormFinder, BestKeeper, and RefFinder) were applied to determine the expression stabilities of these genes in 72 samples at different salt concentrations in different tissues. PP2A and TUA5 were the most stable reference genes in different tissues and salt treatments, whereas DREB1D was the least stable. The two reference genes were sufficient to normalize gene expression across all sample sets. The suitability of identified reference genes was validated with MYB and AP2 in germinating seeds of S. glauca exposed to different NaCl concentrations. Our study provides a foundational framework for standardizing qPCR analyses, enabling accurate gene expression profiling in S. glauca.

Transcriptome analysis to identify key genes involved in terpenoid and rosmarinic acid biosynthesis in lemon balm (Melissa officinalis).

Melissa officinalis (lemon balm) is a well-known pharmaceutical plant in traditional medicine around the world because of the high-value secondary metabolites. Nowadays, advances in computational biology and bioinformatics have opened new avenues to plant-based natural product drug discovery. Despite the pharmacological importance, there is low information about the genes encoding the important biosynthetic pathways related to the secondary metabolite in M. officinalis. In this study, the main genes related to the rosmarinic acid (RA) and terpenoid biosynthesis pathways were detected using transcriptome analysis. Furthermore, we isolated and characterized a novel M. officinalis Hydroxyphenylpyruvate reductase (HPPR) gene involved in RA biosynthesis pathway. An effective pipeline was used to generate 37,055 unigenes by evaluating 42,837,601 Illumina paired-end reads. Functional annotation of the unigenes revealed that 27,363 (73.84%) and 35,822 (96.67%) unigenes had significant similarity to identified proteins in the SwissProt and NR databases, respectively. Also, 10,062 (36.83%) out of 37,055 unigenes were assigned to 399 KEGG pathways. Since terpenes and RA are two prominent metabolites in this plant, the attention of this study has been on the pathways related to them. A total of 149 unigenes were found that are related to the terpenoids biosynthesis, including 75 unigenes involved in the methyl-erythritol phosphate and mevalonate pathway, terpenoid backbone biosynthesis genes, and 74 unigenes related to the terpene synthase. We also identified 144 and 30 unigenes that were associated with the biosynthesis of phenylpropanoid and the rosmarinic acid pathway. Consequently, this investigation can be a comprehensive and accurate transcriptome basis for further investigation in the metabolic engineering and detection of new genes and pathways in M. officinalis.

Hairy Root Culture an Alternative for Bioactive Compound Production from Medicinal Plants.

Medicinal plants produce a diverse group of phytocompounds like anthraquinones, alkaloids, anthocyanins, flavonoids, saponins, and terpenes which are used in pharmaceutical, perfume, cosmetics, dye and flavor industries. Commercial source of these metabolites is field-grown plants, which are generally influenced by seasonal changes. Biotechnology possesses a significant role in production of high-value secondary metabolites. By incorporating biotechnological methods, it is feasible to manage biosynthetic pathways of the plant to enhance phytocompound production that is of pharmaceutical interest. Plant cell suspension, shoot, adventitious root and hairy root culture are considered as alternative methods for important bioactive compound production. These methods are controllable, sustainable and overcome several inconveniences for large scale secondary metabolites production. At present research on hairy root culture for valuable bioactive compound production has gained a lot of attention. Agrobacterium rhizogenes is an agent which causes hairy root disease in a plant and this leads to the neoplastic growth of root which is characterized by higher growth rate and genetic stability. Various studies explore the hairy root culture for production of a wide range of bioactive compounds. Scale-up of hairy root culture using bioreactors has provided an opportunity to enhance bioactive compound production at the commercial level. The present review discusses the role of hairy root culture in the production of valuable bioactive compounds, the effect of culture parameters on bioactive compound production and bioreactor applications.

Enrichment of alliin in different in vitro grown tissues of Allium sativum through CdCl2 elicitation as revealed by high performance thin layer chromatography (HPTLC)

Plant cell tissue and organ culture techniques provide an efficient way for the biosynthesis of high-value secondary metabolites in which elicitation has been recognized to be a potent approach in enriching the bioactive compounds. In this study, an effort was made to assess the influence of Cadmium chloride (CdCl2) on alliin yield of different in vitro grown tissues such as callus, somatic embryos, leaves and roots of Allium sativum. The tissues were cultured in MS amended with various levels of CdCl2 [control (0 mM), T1 (0.05 mM), T2 (0.1 mM), T3 (0.15 mM) and T4 (0.2 mM)] and the yield of alliin was quantified by using high performance thin layer chromatography (HPTLC). Biochemical analyses revealed that sample tissues under increasing CdCl2 conditions accumulated higher levels of sugar, protein and proline with maximum content of sugar (19.61 mg g−1 FW), protein (6.01 mg g−1 FW) and proline (12.19 mg g−1 FW) in leaves at T3 after 4 days of elicitor treatment. Changes within the activities of varied antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) was assayed as the addition of CdCl2 in the medium causes stress. We noted that with an elevated level of CdCl2, antioxidant enzyme activity increased linearly with the highest activity observed in leaves at T4 of 6 days of elicitation (4.97 EU min−1 mg−1 protein CAT, 6.30 EU min−1 mg−1 protein SOD and 1.125 EU min−1 mg−1 protein APX). Increased activities of enzymes are involved in negating the cellular stress triggered due to the overproduction of ROS. The yield of alliin in response to varying levels of CdCl2 was estimated by using a simple, rapid, and sensitive HPTLC method. The best separation was achieved using butanol:propanol:acetic acid:water (3:1:1:1) as mobile phase. For analysis of alliin, the plates were scanned at 550 nm after spraying with ninhydrin and a good resolution was achieved with Rf value of 0.66. In our study, the alliin yield enhanced in all tested cultures with the elicitor level up to T3. In leaves, maximum accumulation of alliin (3.37 μg g−1 dry wt.) was noted on 0.15 mM (T3) CdCl2 added medium. Our data indicated the increase of alliin in CdCl2 added cultures, which suggests that the use of elicitors as a potential strategy for augmenting secondary metabolite synthesis.

Anti-Obesity Attributes; UHPLC-QTOF-MS/MS-Based Metabolite Profiling and Molecular Docking Insights of Taraxacum officinale.

The naturopathic treatment of obesity is a matter of keen interest to develop efficient natural pharmacological routes for disease management with low or negligible toxicity and side effects. For this purpose, optimized ultrasonicated hydroethanolic extracts of Taraxacum officinale were evaluated for antiobesity attributes. The 2,2-diphenyl-1-picrylhydrazyl method was adopted to evaluate antioxidant potential. Porcine pancreatic lipase inhibitory assay was conducted to assess the in vitro antiobesity property. Ultra-high performance chromatography equipped with a mass spectrometer was utilized to profile the secondary metabolites in the most potent extract. The 60% ethanolic extract exhibited highest extract yield (25.05 ± 0.07%), total phenolic contents (123.42 ± 0.007 mg GAE/g DE), total flavonoid contents (55.81 ± 0.004 RE/g DE), DPPH-radical-scavenging activity (IC50 = 81.05 ± 0.96 µg/mL) and pancreatic lipase inhibitory properties (IC50 = 146.49 ± 4.24 µg/mL). The targeted metabolite fingerprinting highlighted the presence of high-value secondary metabolites. Molecular-binding energies computed by docking tool revealed the possible contribution towards pancreatic lipase inhibitory properties of secondary metabolites including myricetin, isomangiferin, icariside B4, kaempferol and luteolin derivatives when compared to the standard drug orlistat. In vivo investigations revealed a positive impact on the lipid profile and obesity biomarkers of obese mice. The study presents Taraxacum officinale as a potent source of functional bioactive ingredients to impart new insights into the existing pool of knowledge of naturopathic approaches towards obesity management.

Phytochemicals and antioxidant capacity of wild growing and in vitro Hypericum heterophyllum

In vitro cultures of Hypericum heterophyllum Vent. was established by using MS and MS-B5 medium contained plant growth regulators such as kinetin, BAP, IAA, TDZ and picloram. Flower, leaf, stem and in vitro samples (callus and in vitro plantlets) of H. heterophyllum were analysed by LC-MS/MS for their chemical contents such as quinic acid, gallic acid, (+)-catechin, ferulic acid, vanillic acid, p-coumaric acid, caffeic acid, and quercetin; moreover their radical scavening activities conducted by DPPH and ABTS methods were evaluated. Among all the analysed samples, the in vitro plantlets shown the highest antioxidant activity (IC50, 220 μg/mL for DPPH and 254 μg/mL for ABTS), probably due to the presence of phenolic acids and flavonoids, specifically the higher total phenolic content (64.4 mg GAE/g extract) than other samples. The phytochemical variation among all samples was discussed through principal component analysis (PCA) and hierarchical cluster analysis (HCA). The in vitro plantlets might offer possibilities for the production of high-value secondary metabolites as pharmaceuticals and food preservatives. This study is the first report on analyses and comparison of secondary metabolites and antioxidant activities in different plant parts and in vitro samples of endemic H. heterophyllum.

Synthetic biology, systems biology, and metabolic engineering of Yarrowia lipolytica toward a sustainable biorefinery platform.

Yarrowia lipolytica is an oleaginous yeast that has been substantially engineered for production of oleochemicals and drop-in transportation fuels. The unique acetyl-CoA/malonyl-CoA supply mode along with the versatile carbon-utilization pathways makes this yeast a superior host to upgrade low-value carbons into high-value secondary metabolites and fatty acid-based chemicals. The expanded synthetic biology toolkits enabled us to explore a large portfolio of specialized metabolism beyond fatty acids and lipid-based chemicals. In this review, we will summarize the recent advances in genetic, omics, and computational tool development that enables us to streamline the genetic or genomic modification for Y. lipolytica. We will also summarize various metabolic engineering strategies to harness the endogenous acetyl-CoA/malonyl-CoA/HMG-CoA pathway for production of complex oleochemicals, polyols, terpenes, polyketides, and commodity chemicals. We envision that Y. lipolytica will be an excellent microbial chassis to expand nature's biosynthetic capacity to produce plant secondary metabolites, industrially relevant oleochemicals, agrochemicals, commodity, and specialty chemicals and empower us to build a sustainable biorefinery platform that contributes to the prosperity of a bio-based economy in the future.

Membrane transporters: the key drivers of transport of secondary metabolites in plants.

This review summarizes the recent updates in the area of transporters of plant secondary metabolites, including their applied aspects in metabolic engineering of economically important secondary metabolites. Plants have evolved biosynthetic pathways to produce structurally diverse secondary metabolites, which serve distinct functions, including defense against pathogens and herbivory, thereby playing a pivotal role in plant ecological interactions. These compounds often display interesting bioactivities and, therefore, have been used as repositories of natural drugs and phytoceuticals for humans. At an elevated level, plant secondary metabolites could be cytotoxic to the plant cell itself; therefore, plants have developed sophisticated mechanisms to sequester these compounds to prevent cytotoxicity. Many of these valuable natural compounds and their precursors are biosynthesized and accumulated at diverse subcellular locations, and few are even transported to sink organs via long-distance transport, implying the involvement of compartmentalization via intra- and intercellular transport mechanisms. The transporter proteins belonging to different families of transporters, especially ATP binding cassette (ABC) and multidrug and toxic compound extrusion (MATE) have been implicated in membrane-mediated transport of certain plant secondary metabolites. Despite increasing reports on the characterization of transporter proteins and their genes, our knowledge about the transporters of several medicinally and economically important plant secondary metabolites is still enigmatic. A comprehensive understanding of the molecular mechanisms underlying the whole route of secondary metabolite transportome, in addition to the biosynthetic pathways, will aid in systematic and targeted metabolic engineering of high-value secondary metabolites. The present review embodies a comprehensive update on the progress made in the elucidation of transporters of secondary metabolites in view of basic and applied aspects of their transport mechanism.

Overexpression of polygalacturonase-inhibiting protein (PGIP) gene from Hypericum perforatum alters expression of multiple defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens in tobacco

Hypericum perforatum L is a remarkable source of high-value secondary metabolites with increasing applications in pharmaceutical industry. However, improvement in the production of secondary metabolites through genetic engineering is a demanding task, as H. perforatum is not amenable to Agrobacterium tumefaciens-mediated transformation. In this study, we identified a Polygalacturonase-inhibiting protein (PGIP) gene from a subtractive cDNA library of A. tumefaciens-treated H. perforatum suspension cells. The role of HpPGIP in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum overexpressing HpPGIP alone or fused at the N-terminus to Phenolic oxidative coupling protein (Hyp-1), a gene that positively modulates resistance to A. tumefaciens. Furthermore, virus-induced gene silencing was employed to knock down the expression of the PGIP homologous in N. benthamiana. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in both HpPGIP and Hyp-1-PGIP transgenic plants, as assessed by GUS staining assays. However, silencing of PGIP in N. benthamiana increased the resistance to A. tumefaciens rather than susceptibility, which correlated with induction of pathogenesis-related proteins (PRs). The expression of core genes involved in several defense pathways was also analyzed in transgenic tobacco plants. Overexpression of HpPGIP led to up-regulation of key genes involved in hormone signaling, microRNA-based gene silencing, homeostasis of reactive oxygen species, and the phenylpropanoid pathway. Overexpression of Hyp-1-PGIP seemed to enhance the effect of PGIP on the expression of most genes analyzed. Moreover, HpPGIP was detected in the cytoplasm, nucleus and the plasma membrane or cell wall by confocal microscopy. Overall, our findings suggest HpPGIP modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.

Transgenic expression of Hyp-1 gene from Hypericum perforatum L. alters expression of defense-related genes and modulates recalcitrance to Agrobacterium tumefaciens.

Phenolic oxidative coupling protein (Hyp-1) isolated from Hypericum perforatum L. was characterized as a defense gene involved in H. perforatum recalcitrance to Agrobacterium tumefaciens-mediated transformation Hypericum perforatum L. is a reservoir of high-value secondary metabolites of increasing interest to researchers and to the pharmaceutical industry. However, improving their production via genetic manipulation is a challenging task, as H. perforatum is recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, phenolic oxidative coupling protein (Hyp-1), a pathogenesis-related (PR) class 10 family gene, was selected from a subtractive cDNA library from A. tumefaciens-treated H. perforatum suspension cells. The role of Hyp-1 in defense against A. tumefaciens was analyzed in transgenic Nicotiana tabacum and Lactuca sativa overexpressing Hyp-1, and in Catharanthus roseus silenced for its homologous Hyp-1 gene, CrIPR. Results showed that Agrobacterium-mediated expression efficiency greatly decreased in Hyp-1 transgenic plants. However, silencing of CrIPR induced CrPR-5 expression and decreased expression efficiency of Agrobacterium. The expression of core genes involved in several defense pathways was also analyzed in Hyp-1 transgenic tobacco plants. Overexpression of Hyp-1 led to an ample down-regulation of key genes involved in auxin signaling, microRNA-based gene silencing, detoxification of reactive oxygen species, phenylpropanoid pathway and PRs. Moreover, Hyp-1 was detected in the nucleus, plasma membrane and the cytoplasm of epidermal cells by confocal microscopy. Overall, our findings suggest Hyp-1 modulates recalcitrance to A. tumefaciens-mediated transformation in H. perforatum.