Although a number of plant natural products are derived from benzoic acid, the biosynthesis of this structurally simple precursor is poorly understood. Hypericum calycinum cell cultures accumulate a benzoic acid-derived xanthone phytoalexin, hyperxanthone E, in response to elicitor treatment. Using a subtracted complementary DNA (cDNA) library and sequence information about conserved coenzyme A (CoA) ligase motifs, a cDNA encoding cinnamate:CoA ligase (CNL) was isolated. This enzyme channels metabolic flux from the general phenylpropanoid pathway into benzenoid metabolism. HcCNL preferred cinnamic acid as a substrate but failed to activate benzoic acid. Enzyme activity was strictly dependent on the presence of Mg²⁺ and K⁺ at optimum concentrations of 2.5 and 100 mm, respectively. Coordinated increases in the Phe ammonia-lyase and HcCNL transcript levels preceded the accumulation of hyperxanthone E in cell cultures of H. calycinum after the addition of the elicitor. HcCNL contained a carboxyl-terminal type 1 peroxisomal targeting signal made up by the tripeptide Ser-Arg-Leu, which directed an amino-terminal reporter fusion to the peroxisomes. Masking the targeting signal by carboxyl-terminal reporter fusion led to cytoplasmic localization. A phylogenetic tree consisted of two evolutionarily distinct clusters. One cluster was formed by CoA ligases related to benzenoid metabolism, including HcCNL. The other cluster comprised 4-coumarate:CoA ligases from spermatophytes, ferns, and mosses, indicating divergence of the two clades prior to the divergence of the higher plant lineages.

Fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple (Malus × domestica). The phytoalexins of apple are biphenyls and dibenzofurans, whose carbon skeleton is formed by biphenyl synthase (BIS), a type III polyketide synthase. In the recently published genome sequence of apple ‘Golden Delicious’, nine BIS genes and four BIS gene fragments were detected. The nine genes fall into four subfamilies, referred to as MdBIS1 to MdBIS4. In a phylogenetic tree, the BIS amino acid sequences from apple and Sorbus aucuparia formed an individual cluster within the clade of the functionally diverse type III polyketide synthases. cDNAs encoding MdBIS1 to MdBIS4 were cloned from fire-blight-infected shoots of apple ‘Holsteiner Cox,’ heterologously expressed in Escherichia coli, and functionally analyzed. Benzoyl-coenzyme A and salicoyl-coenzyme A were the preferred starter substrates. In response to inoculation with E. amylovora, the BIS3 gene was expressed in stems of cv Holsteiner Cox, with highest transcript levels in the transition zone between necrotic and healthy tissues. The transition zone was the accumulation site of biphenyl and dibenzofuran phytoalexins. Leaves contained transcripts for BIS2 but failed to form immunodetectable amounts of BIS protein. In cell cultures of apple ‘Cox Orange,’ expression of the BIS1 to BIS3 genes was observed after the addition of an autoclaved E. amylovora suspension. Using immunofluorescence localization under a confocal laser-scanning microscope, the BIS3 protein in the transition zone of stems was detected in the parenchyma of the bark. Dot-shaped immunofluorescence was confined to the junctions between neighboring cortical parenchyma cells.

Sorbus aucuparia cell cultures accumulate biphenyl and dibenzofuran phytoalexins in response to elicitor treatment. These polyketide derivatives arise from the starter substrate benzoyl-CoA, the biosynthesis of which is largely unresolved. Two CoA ligases involved are cinnamate:CoA ligase and benzoate:CoA ligase, which were assumed to be related in S. aucuparia to the ubiquitous 4-coumarate:CoA ligase (4CL). cDNAs encoding three distinct 4CLs from elicitor-treated S. aucuparia cell cultures were isolated using RT-PCR and RACE techniques and functionally expressed in Escherichia coli as His(6)-tagged proteins (Sa4CL2 and Sa4CL3) or GST-fusion protein (Sa4CL1). All three isoenzymes preferred 4-coumaric acid over cinnamic acid in spectrophotometric assays and failed to utilize benzoic acid in radioisotopic assays. After elicitor treatment of S. aucuparia cell cultures, the transcript levels of all three Sa4CLs increased but were significantly lower than the maximum expression rates of the phenylalanine ammonia-lyase (PAL) and biphenyl synthase 1 (BIS1) genes. The substrate specificities and the expression profiles indicate that the three 4CL isoenzymes are not involved in benzoyl-CoA biosynthesis in S. aucuparia cell cultures. Sa4CL3 and PAL transcripts also accumulated in response to light treatment. Phylogenetically, Sa4CL1 and Sa4CL2 belong to the class I cluster and Sa4CL3 groups in the class II cluster. Sa4CL3 contains a 49-amino acid N-terminal extension, which includes a chloroplast sorting signal.

Cell cultures of Sorbus aucuparia respond to the addition of chitosan with the accumulation of the biphenyl phytoalexin aucuparin. The carbon skeleton of this inducible defense compound is formed by biphenyl synthase (BIS) from benzoyl-CoA and three molecules of malonyl-CoA. The formation of benzoyl-CoA proceeds via benzaldehyde as an intermediate. Benzaldehyde dehydrogenase (BD), which converts benzaldehyde into benzoic acid, was detected in cell-free extracts from S. aucuparia cell cultures. BD and BIS were induced by chitosan treatment. The preferred substrate for BD was benzaldehyde (K_m=49 μM). Cinnamaldehyde and various hydroxybenzaldehydes were relatively poor substrates. BD activity was strictly dependent on the presence of NAD⁺ as a cofactor (K_m=67 μM)

Upon pathogen attack, fruit trees such as apple (Malus spp.) and pear (Pyrus spp.) accumulate biphenyl and dibenzofuran phytoalexins, with aucuparin as a major biphenyl compound. 4-Hydroxylation of the biphenyl scaffold, formed by biphenyl synthase (BIS), is catalyzed by a cytochrome P450 (CYP). The biphenyl 4-hydroxylase (B4H) coding sequence of rowan (Sorbus aucuparia) was isolated and functionally expressed in yeast (Saccharomyces cerevisiae). SaB4H was named CYP736A107. No catalytic function of CYP736 was known previously. SaB4H exhibited absolute specificity for 3-hydroxy-5-methoxybiphenyl. In rowan cell cultures treated with elicitor from the scab fungus, transient increases in the SaB4H, SaBIS, and phenylalanine ammonia lyase transcript levels preceded phytoalexin accumulation. Transient expression of a carboxyl-terminal reporter gene construct directed SaB4H to the endoplasmic reticulum. A construct lacking the amino-terminal leader and transmembrane domain caused cytoplasmic localization. Functional B4H coding sequences were also isolated from two apple (Malus × domestica) cultivars. The MdB4Hs were named CYP736A163. When stems of cv Golden Delicious were infected with the fire blight bacterium, highest MdB4H transcript levels were observed in the transition zone. In a phylogenetic tree, the three B4Hs were closest to coniferaldehyde 5-hydroxylases involved in lignin biosynthesis, suggesting a common ancestor. Coniferaldehyde and related compounds were not converted by SaB4H.

A simple, precise, rapid and accurate isocratic reversed-phase high-performance liquid chromatographic method was developed and validated for the simultaneous determination of biphenyl (aucuparin and noraucuparin) and dibenzofuran (eriobofuran) phytoalexin from elicitor treated cell culture of Sorbus aucuparia (mountain ash). These phytoalexins play crucial role in combating scab disease in many commercially important rosaceous plants, such as apple, pear and mountain ash. The isocratic separation was performed in a Luna C₁₈ reversed-phase column (250 × 4.6 mm, 5 μm particle size) using a mobile phase of 1 mM trifluoroacetic acid (TFA) in water with methanol [40:60 (v/v)]. Quantization of phytoalexin was carried out on Shimadzu-HPLC system using a Photo Diode Array (PDA) detector at 254 nm by comparing the peak area. Peak purity and identity were confirmed by UV spectroscopy and ESI–MS-MS in the negative ion mode. The different analytical performance parameters such as linearity, accuracy, precision, limit of detection and limit of quantification were determined according to the International Conference on Harmonization guidelines. Linearity was observed in the concentration range of 3–400 µg/mL with excellent correlation coefficient (R² ≥ 0.995). This newly developed method is rapid, easy, cost-effective and can be used for monitoring scab-resistance potential of rosaceous plants.

Monoamine oxidase inhibition is an important therapeutic approach for various neurodegenerative disorders.
Reversible MAO inhibitors selectively targeting only one isoform possess substantial merit in
terms of safety, efficacy, and side effect profile. This study aimed to isolate the secondary metabolites
of Zanthoxylum flavum stems and evaluate their recombinant human MAO inhibition, antimicrobial,
and antiprotozoal activities. As a result, fourteen compounds were isolated and identified (nine of them
were reported from Z. flavum for the first time). Compound 3 (sesamin) exhibited potent selective MAO-B
inhibition (IC50 value of 1.45 ± 0.05 mM) which reported herein for the first time. Compound 2 showed
selective MAO-A inhibition activity, compound 5 exhibited good trypanocidal activity, and compound
7 displayed moderate antibacterial activity. The promising MAO-B inhibitory activity of sesamin provoked
us to further explore the kinetic properties, the binding mode, and the underlying mechanism
of MAO-B inhibition by this lignan. This detailed investigation substantiated a reversible binding and
mixed MAO-B catalytic function inhibition via sesamin (Ki: 0.473 ± 0.076 lM). Selectivity and reversibility
of sesamin on MAO-B provide exciting prerequisites for further in vivo investigation to confirm its
therapeutic potentiality.

Medicinal plants are sustainable bio-factories for valuable active pharmaceutical ingredients (API). They are commonly grown in the field and their extracts have a given combination of constituents. There is some variation due to climate fluctuations and plant diseases (microbial infections), genotypic changes, soil differences, etc. Additionally, fertile agricultural areas are increasingly limited. However, these variations are undesired because they are non-controllable and can affect the batch conformity of a drug significantly. This is a challenge for producers of phyto-pharmaceuticals, and the variations in the API composition are compensated by mixing extracts from various batches to achieve the required continuous quality of an authorized drug. These drawbacks of field cultivation are overcome by well-defined bioreactor-based cultivation. Biomass growth and API production take place under variable but controllable cultivation conditions, resulting in customized extracts. Variation of the cultivation conditions leads to qualitative and/or quantitative changes in the metabolome. During bioreactor cultivation, plant cells tend to stay connected after division, which leads to the formation of aggregates. The size of shear-sensitive plant cell aggregates influenced by hydrodynamic forces resulting from mechanical agitation was often recognized as an intangible parameter, which might be responsible for general variability in plant cell culture processes. To date, however, the bioreactor approach is not often industrially implemented. This chapter provides an overview of the challenges in the cultivation of plant cell systems, briefly illustrated by (i) research on Hypericum perforatum tissue cultures into up-to-date approaches for production of hyperforin and hypericin, possibly functional at a pre-commercial level in the future, and (ii) effects of hydrodynamic mechanical forces on Taxus chinensis submerged cultures for production of paclitaxel.

Papaya fruits have great nutritional and economical values. The ripening stage of papaya fruits at the time of harvesting massively impacts the quality and shelf-life of fruits. Volatile organic compounds (VOCs) emitting from the papaya fruits during the ripening process could be used as a real-time non-invasive biomarker to characterize the ripening stage. This paper reports identification of VOCs as non-invasive ripening biomarkers of papaya fruits based on solid-phase micro-extraction coupled with gas chromatography–mass spectrometry (GC–MS) analysis of VOCs emitted from the papaya fruit cv. ‘Red Lady’. Three ripening stages were studied, viz., green unripe (UR), yellowish-green intermediate ripe (IR), and yellow full ripe (FR). GC–MS analyses and the subsequent statistical studies identified a total of 35 VOCs. Partial Least Squares Discriminant Analysis (PLS-DA) of VOCs from the three ripening stages identified six biomarker VOCs, which can efficiently distinguish between ripening stages. Among the six ripening biomarkers, three VOCs (methyl hexanoate, 3-carene and longifolene) showed a remarkable correlation with the ripening-associated changes in the fruit nutritional profile. The biomarkers reported here could be used as a viable technology for non-invasive monitoring of ripening stages and the nutritional value of papaya fruits.