Cell Wall Damage-Induced Lignin Biosynthesis Is Regulated by a Reactive Oxygen Species- and Jasmonic Acid-Dependent Process in Arabidopsis

The plant cell wall is a dynamic and complex structure whose functional integrity is constantly being monitored and maintained during development and interactions with the environment. In response to cell wall damage (CWD), putatively compensatory responses, such as lignin production, are initiated. In this context, lignin deposition could reinforce the cell wall to maintain functional integrity. Lignin is important for the plant’s response to environmental stress, for reinforcement during secondary cell wall formation, and for long-distance water transport. Here, we identify two stages and several components of a genetic network that regulate CWD-induced lignin production in Arabidopsis (Arabidopsis thaliana). During the early stage, calcium and diphenyleneiodonium-sensitive reactive oxygen species (ROS) production are required to induce a secondary ROS burst and jasmonic acid (JA) accumulation. During the second stage, ROS derived from the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D and JA-isoleucine generated by JASMONIC ACID RESISTANT1, form a negative feedback loop that can repress each other’s production. This feedback loop in turn seems to influence lignin accumulation. Our results characterize a genetic network enabling plants to regulate lignin biosynthesis in response to CWD through dynamic interactions between JA and ROS

TaReCa – Cascade utilization of horticultural biomass for a resource efficient production of valuable bioactive substances

Viele pflanzliche Sekundärmetabolite haben antioxidative oder andere bioaktive Eigenschaften, weshalb sie einerseits wichtige Bestandteile der menschlichen Ernährung sind, andererseits aber auch als pharmazeutische Verbindungen oder als Substrat für die chemische Synthese von bioaktiven Substanzen verwendet werden. Pflanzen induzieren die Produktion solcher nutzbaren Sekundärmetabolite wie z.B. Flavonoiden als Reaktion auf abiotischen Stress. Die Produktion von Gemüse und Früchten in Gewächshäusern hinterlässt große Mengen an ungenutzter pflanzlicher Biomasse, welche eine potentielle Ressource für die Gewinnung wertvoller Metabolite darstellt. Durch eine kaskadenartige Verwendung von Gartenbaukulturen zur Produktion von Früchten und Gemüse mit einer anschließenden Gewinnung hochwertiger Substanzen aus der verbleibenden Restbiomasse würde ein erheblicher Mehrwert generiert. Das Projekt TaReCa bearbeitet die Entwicklung einer maßgeschneiderten Kaskadenverwertung von Paprikapflanzen-Restbiomasse aus dem Gartenbau. Dabei soll der pflanzliche Sekundärmetabolismus durch spezifische abiotische Stressbedingungen nach der Fruchternte gezielt induziert werden, um die Konzentrationen der Zielmetaboliten zu steigern. Durch umweltfreundliche und wirtschaftliche Extraktionsprozesse und eine anschließende Verwertung des verbleibenden Pflanzenmaterials in einer Bioraffinerie wird die Wertschöpfungskette erweitert. Eine Analyse der Anwendungsgebiete sowie Untersuchungen zur Akzeptanz der induzierten Inhaltsstoffe, Prozesse und Technologien werden helfen, das Marktpotenzial der Restbiomasse für die Nutzung in Kaskaden zu evaluieren. Die maßgeschneiderte Nutzung von Gartenbaubiomasse durch Lebensmittelproduktion, Extraktion bioaktiver Sekundärmetabolite und Bioraffinerien kann wirtschaftlich relevante, biobasierte Produkte für industrielle Anwendungen erzeugen und somit zur Entwicklung einer nachhaltigen, effizienten und integrierten Bioökonomie beitragen, ohne mit der Lebensmittelproduktion zu konkurrieren.Many plant secondary metabolites have antioxidant or pharmaceutically relevant properties, which makes them important components of the human diet, but also as pharmaceutical compounds or for the chemical synthesis of bioactive substances. Plants induce the production of secondary metabolites, e.g. flavonoids in response to environmental stress stimuli. The production of vegetables and fruits in greenhouses leaves huge amounts of so far under-utilized biomass after fruit harvest, which is a potential source for production of valuable metabolites. A cascade utilization of horticultural crops to produce fruits and vegetables with subsequent extraction of high quality compounds would generate significant added value. The project TaReCa is working on the development of a tailored cascade utilization of bell pepper plant residues from horticulture. The secondary metabolism will be induced by specific abiotic stress treatments after the last fruit harvest, in order to increase the concentrations of the target metabolites. Eco-friendly and economical extraction processes and subsequent utilization of the remaining plant material in a biorefinery will expand the value chain. An analysis of the application areas as well as studies on the acceptance of the induced ingredients, processes and technologies will help to evaluate the market potential of the residual biomass for the proposed cascaded use. The tailored utilization of horticultural biomass in food production, extraction of bioactive secondary metabolites and biorefineries can produce economically relevant bio-based products for industrial applications and thus contribute to the development of a sustainable, efficient and integrated bioeconomy without competing with food production

Color for Life: Biosynthesis and Distribution of Phenolic Compounds in Pepper (<i>Capsicum annuum</i>)

Fruits and vegetables are an important supplier of biological active substances, such as vitamins and secondary metabolites (SM) for human nutrition, but also for further industrial applications. Pepper (Capsicum annuum) is grown and consumed all over the world as a fresh vegetable or dried as a spice. It is also used as a coloring agent, as well for medical purposes. Pepper fruits are considered as an attractive source of health-related compounds, but other organs like the leaves and stem also contain considerable amounts of antioxidants, e.g., phenolic compounds. This indicates potential for valorization of residual biomass from horticultural production by using innovative bioeconomic concepts. Herein, we present an overview about the biosynthesis of phenolic compounds, with a special focus on flavonoids and their regulation in pepper, the current knowledge of amounts and distribution of these valuable substances, as well as possible strategies for: (1) increasing flavonoid contents in pepper, (2) improving the nutritional value of fruits, and (3) new concepts for utilization of residual biomass from horticultural production

Application of abiotic stresses induce the production of valuable specialized metabolites in bell pepper leaves

At the end of annual horticultural production of food large amounts of unused green plant material are left behind. This residual plant biomass contains lignocellulosic material as well as plant specialized metabolites which can be of use for industrial purposes like for example additives for food or cosmetics. The interdisciplinary project TaReCa aims at utilizing targeted abiotic stress treatments to induce increased accumulation of valuable flavonoids in green plant biomass of bell pepper after the last fruit harvest in commercial greenhouses for extraction and industrial utilization.To identify suitable abiotic stresses for induction of flavonoids, different intensities and durations of salt and cold stress treatments were applied to young bell pepper plants. With a combination of salt and cold stress, we were able to increase total phenolics and flavonoids contents 2- and 4-fold in leaves of young bell pepper plants of two commercially grown lines (cv. Mazurka, cv. Stayer). In addition, the project´s target flavonoid metabolites graveobioside A and cynaroside were strongly accumulated in both lines, leading to a 3- and 13-fold induction in leaves of cv. Mazurka. Phenotyping methods based on leaf color and assessment of leaf fluorescence were used to monitor plant stress responses and to develop an easy-to-use method to predict changes in leaf flavonoid or target metabolite content in production greenhouses. This novel approach of valorization of horticultural side-streams has the potential to add value to the bell pepper production

Targeted application of abiotic stress as a tool to trigger biosynthesis of bioactive flavonoids in Capsicum leaves

Abiotic stresses are usually considered as limiting factors in the horticultural production of bell pepper. However, the enhancing effect of abiotic stress on the biosynthesis of plant secondary metabolites bears the potential for production of valuable bioactive plant compounds via targeted application of abiotic stress. At the end of the annual production cycle of bell pepper, high amounts of residual leaf material remain in the greenhouse. Since bell pepper leaves are known to contain pharmaceutically interesting compounds like the flavonoids cynaroside and graveobioside A (Ellenberger et al., 2020), the residual foliar plant material represents a potential source for valuable metabolites, which could be extracted following the horticultural production of fruits. However, the concentration of those metabolites is often too low for an economically feasible extraction. Therefore, the interdisciplinary project TaReCa aimed at increasing the biosynthesis of those valuable metabolites via application of abiotic stress.To identify suitable abiotic stresses strongly triggering the accumulation of the target flavonoids cynaroside and graveobioside A, various stress treatments (e.g. salinity, temperature stress, UV stress, nitrogen deficiency) were applied in different durations, intensities, and combinations to young bell pepper and chilli plants. Throughout treatments, stress responses of plants were monitored noninvasively using phenotyping methods based on leaf colour, fluorescence, and reflectance to develop an easy-to-use method to predict changes in the leaf content of target flavonoids in production greenhouses. With a combination of salt and moderate cold stress, we were able to increase the target metabolites graveobioside A and cynaroside 3.6- and 23-fold in bell pepper leaves of young plants reaching comparable or even higher concentrations than those described for plants commonly used as sources for these metabolites. This novel approach of valorization of horticultural plant residuals has the potential to add value to the bell pepper production

Targeted induction of plant secondary metabolism in horticultural plants by controlled stress applications

Plants rely on morphological or biochemical mechanisms to protect themselves under challenging environmental conditions. This involves the accumulation of diverse bioactive secondary metabolites. Such secondary metabolites are recognized as valuable compounds for human health, as ingredients of cosmetics or for further industrial purposes, utilized after extraction of purified compounds or in plant extracts. The interdisciplinary projects InducTomE and TaReCa evaluate a novel process to make use of secondary metabolites in residual plant biomass of horticultural plants. In this process, stress treatments after the last fruit harvest are applied to increase the amount of valuable metabolites in the plant residuals. Thereby, the residual biomass can be used for the extraction of industrially relevant metabolites. In a pre-screen, young bell pepper and tomato plants were exposed to various abiotic stress treatments like water or nutrient deficiency, salt stress or cold. We identified suitable stress treatments that induced the accumulation of total phenolics, flavonoids and also valuable target metabolites, like the flavonoid rutin and the polyisoprenoid solanesol. A comparison of commercial lines and a wild relative in these experiments revealed differences in stress-induced metabolite accumulation and biosynthesis gene expression indicating genetic variability of responses of secondary metabolism to stress. According to the results of the pre-screen, stress treatment protocols for the induction of target metabolites will be developed for use in commercial greenhouses. In addition, phenotyping methods were applied to quantify plant stress responses and to monitor the intensity of the treatments. This will also help to develop easy-to-use tools to control the targeted stress application in commercial greenhouses. The proposed method for a targeted tailoring of the secondary metabolism in horticultural residuals demonstrates the valorization of underutilized by-products of horticultural food production. Thereby, it has thus the potential to generate added value and increase sustainability of the horticultural food production

InducTomE – Induction of secondary metabolites in tomato leaves

Plant secondary metabolites are essential components of the human diet, serve as high-value fine chemicals and are utilized as phytomedicines and industrial raw materials. Chemically, secondary metabolites exhibit an enormous diversity and complexity which makes their industrial chemical synthesis difficult and expensive. Therefore, they are often extracted from plants which are grown especially for this purpose, like medicinal plants. On the other hand, large quantities of plant biomass are produced as by-products of horticultural food crop production. As plants increase the production of secondary metabolites in response to abiotic stress, post-harvest treatments of greenhouse-grown crop plants might induce the accumulation of secondary metabolites in these by-products. The identification of suitable stress treatments and extraction procedures would enhance the concentration of secondary metabolites in the green biomass of commercially grown crops and allow for a profitable extraction. The utilization of such by-products for extraction of secondary metabolites would thus add extra value to horticultural crop plant production.The project InducTomE (http://www.biosc.de/inductome) studies the induction of secondary metabolites, namely solanesol and rutin, in tomato by-products, and develops a conceptual process design including extraction procedures and evaluation of the emerging value chains and their economic feasibility. As part of the project, our group evaluates the impact of abiotic stress treatments on the accumulation of secondary metabolites in tomato leaves. We aim to identify abiotic stress conditions which maximize the induction of these target metabolites and can also be cost-efficiently applied to commercially grown mature tomato plants.One-month-old plants of different lines of commercially grown tomato lines in comparison to their wild ancestor, Solanum pennellii, are subjected to different abiotic stress treatments in climate chambers. The content analysis of solanesol and rutin by liquid chromatography-mass spectrometry (LC-MS) is combined with qPCR analyses of key genes involved in their biosynthetic pathways. In addition, plant growth and stress responses are captured by using plant phenotyping imaging techniques. First experiments compared the response of the commercially used tomato line Lyterno and the wild S. pennellii to nitrogen deficiency and drought. Leaf samples were analysed after stress and recovery from nitrogen deficiency and drought. Further experiments will also include cold and light treatments and combinations of nitrogen deficiency, drought, cold and light treatments. When the most suitable abiotic stress treatment is identified, the biosynthetic pathways for solanesol and rutin will be investigated, and plants will be screened for further secondary metabolites which might be of interest for commercial applications.Based on our results, our project partners will apply the most suitable stress treatment to greenhouse grown tomato plants, in order to optimize yield in plants grown under conditions used in horticultural food crop production. Process engineers will develop a technical separation process for the extraction of the target metabolites from green tomato residues. Moreover, the production and capital costs of the downstream processing and the emerging value chains and market potentials will be analysed

Overexpression of a pectin methylesterase inhibitor in Arabidopsis thaliana leads to altered growth morphology of the stem and defective organ separation

The methylesterification status of cell wall pectins, mediated through the interplay of pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs), influences the biophysical properties of plant cell walls. We found that the overexpression of a PMEI gene in Arabidopsis thaliana plants caused the stems to develop twists and loops, most strongly around points on the stem where leaves or inflorescences failed to separate from the main stem. Altered elasticity of the stem, underdevelopment of the leaf cuticle, and changes in the sugar composition of the cell walls of stems were evident in the PMEI overexpression lines. We discuss the mechanisms that potentially underlie the aberrant growth phenotypes

Tailoring of plant secondary metabolism in horticultural plant residuals by targeted stress application

Session 9. Valorization of bio-based industrial side streamsThe production of vegetables and fruits in greenhouses leaves large amounts of residual plant biomass behind, which represents a potential resource for the extraction of valuable secondary metabolites such as flavonoids. Various abiotic stresses induce selectively the biosynthesis of different secondary metabolites in plants. An application of targeted stress treatments in production greenhouses after the last fruit harvest could therefore increase the concentration of specific secondary metabolites in residual plants. Thereby, residual horticultural plant biomass is valorized, as it could be used for the extraction of valuable metabolites. In order to identify stress conditions that induce the accumulation of different valuable secondary metabolites, we measured the content of secondary metabolites of young tomato and bell pepper plants subjected to various abiotic stress treatments. In the projects InducTomE and TaReCa, we investigated the inducibility of selected flavonoids which are of interest due to their antioxidant potential and their bioactive properties, and of the polyisoprenoid solanesol. Additionally, metabolite screenings will be performed to discover further target metabolites of high value and industrial relevance. Thereby, we unfold the full potential of the plant residuals as source of secondary metabolites. To facilitate the implementation of stress treatment protocols in greenhouses and to allow a quality control of the valorized plant biomass, easy-to-use plant phenotyping technologies for the quantification of plant stress responses will be developed. In collaboration with project partners working on gene expression and metabolite analyses, transfer of induction protocols to commercial-like greenhouses, efficient extraction technologies, and an economic evaluation of the secondary metabolites extracted from residual plant biomass, we will demonstrate a proof-of-concept for the proposed valorization process. The proposed valorization and utilization of plant residuals for the extraction of valuable secondary metabolites will contribute to an increased sustainability in horticultural food production