Short rotation coppices (SRCs) represent an important source of biomass. Since they are grown in various mixtures, SRCs represent an excellent opportunity for assessing the effects of local plant neighbourhoods on their performance. We used a common garden experiment consisting of plots that varied in genotype diversity of SRC willows to test for the effects of chemical traits of individual plants and chemical variation in the plots where they grew on insect herbivory. We also explored whether the composition of willows planted in a plot affected their chemistry. To do this, we performed untargeted metabolomics and quantified various chemical traits related to the total set of metabolites we detected, flavonoids, and salicinoids in four willow genotypes. We measured the leaf herbivory that the plants suffered. The genotypes differed in most chemical traits, yet we found only limited effects of individual traits on herbivory damage. Instead, herbivory damage was positively correlated with structural variation in salicinoids in a plot. When analysing the effects of plot chemical variation on herbivory damage separately for each genotype, we found both positive and negative correlations between the two, suggesting both associational resistance and susceptibility. Finally, we also observed a significant effect of the interaction between genotype and plot composition on structural variation in plant chemistry. Overall, our results suggest that high chemical variation in mixed willow SRCs does not necessarily lower the herbivory damage, possibly due to spillover effects of insect herbivores among genotypes. Our results also show that different genotypes respond differently to plot composition in terms of herbivory damage and chemical composition, which may affect their suitability for growing in mixed stands.

The adaptation of pathogenic fungi to plant-specialized metabolites is necessary for their survival and reproduction. The biotrophic fungus Ustilago maydis can cause maize smut and produce tumors in maize (Zea mays), resulting in reduced maize yield and significant economic losses. Qualitative analysis using UPLC-MS/MS revealed that the infection of maize variety B73 with U. maydis resulted in increased levels of phytohormones, phenolics, and alkaloids in maize seedling tissues. However, correlation analysis showed that nearly all compounds in the mechanical damage group were significantly negatively correlated with the shoot growth indexes of maize B73. The correlation coefficients of 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one (HMBOA) and maize B73 shoot length and shoot weight were r = -0.56 (p < 0.01) and r = -0.75 (p < 0.001), respectively. In the inoculation group, these correlations weakened, with the correlation coefficients between HMBOA and maize B73 shoot length and shoot weight being r = 0.02 and r = -0.1, respectively. The correlation coefficients between 6-methoxy-2-benzoxazolinone (MBOA) and the shoot weight were r = -0.73 (p < 0.001) and r = -0.15 in the mechanical damage group and inoculation group, respectively. These findings suggest that increased concentrations of these compounds are more positively associated with mechanical damage than with U. maydis infection. At high concentrations, most of these compounds had an inhibitory effect on U. maydis. This study investigated the ability of U. maydis to regulate various compounds, including phytohormones, phenolic acids, and alkaloids in maize B73, providing evidence that U. maydis has adapted to the specialized metabolites produced by maize B73.

Rice (Oryza sativa L.) husk harbors a substantial proportion of biological metabolites, as one of the most plentiful agriculture by-products in rice milling process, rice husk remains poorly utilized. As a continuing search for potential bioactive molecules from the husk of rice, a totally of twelve conponents (1-12), including six sterol ferulates (1-6), one flavonoid (7), one dipeptide (8), and four phenylpropanoid derivatives (9-12) were obtained. All the chemical structures were elucidated based on comprehensive spectroscopic data. Wherein, compounds 1 and 2 were yield as previous undescribed metabolites, and the comprehensive NMR data for compounds 3 and 4 were first presented in its entirety. Motivated by the similarity of the structural motifs of components 1-6 to that of reported sterol ferulates, the antioxidant and anti-inflammatory effects for compounds 1-6 were evaluated in vitro. Among them, compounds 5/6 had a significant antioxidant activity compare to that of vitamin E in both DPPH and reducing power assay up to the concentration 40 μg/ml; while compounds 1 and 2 exhibited weak suppressive effect on the production of nitric oxide, with the IC50 values of 53.27 ± 1.37 μM.

The recent biological invasion of box tree moth Cydalima perspectalis on Buxus trees has a major impact on European boxwood stands through severe defoliation. This can hinder further regrowth and threaten survival of populations. In a mesocosm approach and controlled larval density over a 2-month period, responses of B. sempervirens essential and specialized metabolites were characterized using metabolomics, combining 1H-NMR and LC-MS/MS approaches. This is the first metabolome depiction of major Buxus responses to boxwood moth invasion. Under severe predation, remaining green leaves accumulate free amino acids (with the noticeable exception of proline). The leaf trans-4-hydroxystachydrine and stachydrine reached 10-13% and 2-3% (DW), while root content was lower but also modulated by predation level. Larval predation promoted triterpenoid and (steroidal) alkaloid synthesis and diversification, while flavonoids did not seem to have a relevant role in Buxus resistance. Our results reveal the concomitant responses of central and specialized metabolism, in relation to severity of predation. They also confirm the potential of metabolic profiling using 1H-NMR and LC-MS to detect re-orchestration of metabolism of native boxwood after severe herbivorous predation by the invasive box-tree moth, and thus their relevance for plant-insect relationships and ecometabolomics.

For centuries plants have been intensively utilized as reliable sources of food, flavoring, and pharmaceutical ingredients. However, plant natural habitats are being rapidly lost due to the climate change and agriculture. Plant biotechnology offers a sustainable approach for the bioproduction of specialized plant metabolites. The unique structural features of plant-derived specialized metabolites, such as their safety profile and multi-target spectrum, have led to the establishment of many plant-derived drugs. However, there are still many challenges to overcome regarding the production of these metabolites from plant in vitro systems and establish a sustainable large-scale biotechnological process. These challenges are due to the peculiarities of plant cell metabolism, the complexity of plant specialized metabolite pathways, and the correct selection of bioreactor systems and bioprocess optimization. In this book chapter, we attempted to focus on the advantages of plant in vitro systems and in particular plant cell suspensions for their cultivation as a source of plant-derived specialized metabolites. A state-of-the-art technological platform for plant cell suspension cultivation from callus induction to lab-scale cultivation, extraction, and purification is presented. Possibilities for bioreactor cultivation of plant cell suspensions in benchtop and large-scale volumes are highlighted, including several examples and patents for industrial production of specialized metabolites.

The insecticidal compound pyrethrin is synthesized in Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevis.) Sch.Bip.; Asteraceae), a plant species endemic to the eastern Mediterranean. Pyrethrin is a mixture of six compounds, pyrethrin I and II, cinerin I and II, and jasmolin I and II. For this study we sampled 15 natural Dalmatian pyrethrum populations covering the entire natural distribution range of the species; Croatian coastal regions and the islands, inland Bosnia and Herzegovina and Montenegro. The plants were grown in a field experiment under uniform growing conditions to exclude a short-term response to environmental factors and instead observe variation in pyrethrin content and composition among and within populations due to genetic adaptation to the native environment. The drivers of local adaptation were explored by examining the role of bioclimatic factors as a cause of population differentiation. Pyrethrins were extracted by ultrasound-assisted extraction, and the extracts were analyzed by HPLC-UV-DAD. The populations differed significantly in the content and composition of pyrethrins. The highest levels of total pyrethrins (1.27% flower DW), were found in population P14 Budva and the significantly highest levels of pyrethrin I in population P14 Vranjske Njive, Podgorica (66.47% of total pyrethrin). Based on bioclimatic conditions of the sampling sites, populations were grouped into five bioclimatic groups (A, B, C, D, and E), which showed qualitative and quantitative variability in pyrethrin content. The most abundant bioclimatic group was bioclimatic group E, which was characterized by the highest average values for pyrethrin I (53.87% of total pyrethrin), total pyrethrin content (1.06% flower DW) and the ratio of pyrethrin I and II (1.85). The correlation analysis between the pyrethrin compounds and some of the bioclimatic variables (e. g., BIO03 Isothermality and BIO04 Temperature seasonality) showed their significant contribution in explaining the variation of pyrethrins in T. cinerariifolium. The differences in pyrethrin content and composition may be partly due to genetic adaptation to the ecological conditions of the native environment. The obtained data would enable the selection of source populations for breeding programs aimed at producing cultivars with desirable biochemical properties and adaptation to different bioclimatic conditions.

Lichens have great potential as food, functional food additives or flavourings. The presence of specific substances with multiple biological activities is one of the characteristics of lichens. However, research on lichens as a food source or functional food additive is limited. The present study simulated, for the first time, the potential bioaccessibility of active compounds from 6 lichen species in simulated gastric and intestinal conditions. An in vitro digestion showed that the lichen substances had different bioaccessibility and stability during digestion. It was found that the application of some metabolic modulators significantly altered the accumulation of metabolites in most species. In addition, the study demonstrated the antimicrobial activity of the tested extracts as well as of 14 isolated lichen metabolites. These multi-directional studies demonstrate the potential of lichens in terms of their use as antimicrobial functional food additives.

Steroidal glycoalkaloids (SGAs) are specialized metabolites produced by hundreds of Solanum species, including important vegetable crops such as tomato, potato, and eggplant. Although it has been known that SGAs play important roles in defense in plants and \"anti-nutritional\" effects (e.g., toxicity and bitterness) to humans, many of these molecules have documented anti-cancer, anti-microbial, anti-inflammatory, anti-viral, and anti-pyretic activities. Among these, α-solasonine and α-solamargine isolated from black nightshade (Solanum nigrum) are reported to have potent anti-tumor, anti-proliferative, and anti-inflammatory activities. Notably, α-solasonine and α-solamargine, along with the core steroidal aglycone solasodine, are the most widespread SGAs produced among the Solanum plants. However, it is still unknown how plants synthesize these bioactive steroidal molecules. Through comparative metabolomic-transcriptome-guided approach, biosynthetic logic, combinatorial expression in Nicotiana benthamiana, and functional recombinant enzyme assays, here we report the discovery of 12 enzymes from S. nigrum that converts the starting cholesterol precursor to solasodine aglycone, and the downstream α-solasonine, α-solamargine, and malonyl-solamargine SGA products. We further identified six enzymes from cultivated eggplant that catalyze the production of α-solasonine, α-solamargine, and malonyl-solamargine SGAs from solasodine aglycone via glycosylation and atypical malonylation decorations. Our work provides the gene tool box and platform for engineering the production of high-value, steroidal bioactive molecules in heterologous hosts using synthetic biology.

Agriculture and global food security encounter significant challenges due to viral threats. In the following decades, several molecular studies have focused on discovering biosynthetic pathways of numerous defensive and signaling compounds, as key regulators of plant interactions, either with viruses or their associated vectors. Nevertheless, the complexities of specialized metabolites mediated plant-virus-vector tripartite viewpoint and the identification of their co-evolutionary crossroads toward antiviral defense system, remain elusive. The current study reviews the various roles of plant-specialized metabolites (PSMs) and how plants use these metabolites to defend against viruses. It discusses recent examples of specialized metabolites that have broad-spectrum antiviral properties. Additionally, the study presents the co-evolutionary basis of metabolite-mediated plant-virus-insect interactions as a potential bioinspired approach to combat viral threats. The prospects also show promising metabolic engineering strategies aimed at discovering a wide range of PSMs that are effective in fending off viruses and their related vectors. These advances in understanding the potential role of PSMs in plant-virus interactions not only serve as a cornerstone for developing plant antiviral systems, but also highlight essential principles of biological control.

Plants employ diverse anti-herbivore defences that can covary to form syndromes consisting of multiple traits. Such syndromes are hypothesized to impact herbivores more than individual defences. We studied 16 species of lowland willows occurring in central Europe and explored if their chemical and physical traits form detectable syndromes. We tested for phylogenetic trends in the syndromes and explored whether three herbivore guilds (i.e., generalist leaf-chewers, specialist leaf-chewers, and gallers) are affected more by the detected syndromes or individual traits. The recovered syndromes showed low phylogenetic signal and were mainly defined by investment in concentration, richness, or uniqueness of structurally related phenolic metabolites. Resource acquisition traits or inducible volatile organic compounds exhibited a limited correlation with the syndromes. Individual traits composing the syndromes showed various correlations to the assemblages of herbivores from the three studied guilds. In turn, we found some support for the hypothesis that defence syndromes are composed of traits that provide defence against various herbivores. However, individual traits rather than trait syndromes explained more variation for all studied herbivore assemblages. The detected negative correlations between various phenolics suggest that investment trade-offs may occur primarily among plant metabolites with shared metabolic pathways that may compete for their precursors. Moreover, several traits characterizing the recovered syndromes play additional roles in willows other than defence from herbivory. Taken together, our findings suggest that the detected syndromes did not solely evolve as an anti-herbivore defence.