To Örebro University

Plant systems have gained increased attention as an alternative platform for producing heterologous proteins, particularly for industrially relevant proteins. The Cynara cardunculus L. flower extract is traditionally used in cheese production across Mediterranean countries due to its milk-clotting properties. To address the growing demand for plant-based milk-clotting enzymes, we investigated the heterologous production of cyprosin B (CYPB), a key milk-clotting enzyme, in Nicotiana benthamiana. We also examined the role of its plant-specific insert (PSI) domain in enzymatic activity, protein yield, and subcellular localization. Full-length CYPB and a PSI domain-deleted variant (CYPBΔPSI) were transiently expressed in N. benthamiana leaves using agroinfiltration. Proteins were purified nine days post-infiltration, yielding ~81 mg/kg (CYPB) and ~60 mg/kg (CYPBΔPSI) fresh weight. CYPBΔPSI showed higher proteolytic activity (~168 IU/mg) than CYPB (~57 IU/mg) and exhibited faster milk clotting times, suggesting that PSI removal may contribute to enhanced enzymatic efficiency. However, additional factors such as altered glycosylation or localization may also play a role. Subcellular localization indicated that CYPB and its PSI domain targeted the vacuole and endocytic vesicles, while CYPBΔPSI predominantly localized to the endoplasmic reticulum and tonoplast. This suggests that the PSI domain’s vital role in vacuolar targeting and membrane permeabilization ultimately influences protein yield. Our study shows N. benthamiana as a scalable platform for producing recombinant CYPB variants with improved enzymatic activity. It highlights the PSI domain's role in vacuolar sorting without impairing 21 function. These findings contribute to the development of plant-based systems for milk-clotting 22 enzymes for cheese-making.

This study determines the functional role of the plant ultraviolet-B radiation (UV-B) photoreceptor, UV RESISTANCE LOCUS 8 (UVR8) under natural conditions using a large-scale 'synchronized-genetic-perturbation-field-experiment'. Laboratory experiments have demonstrated a role for UVR8 in UV-B responses but do not reflect the complexity of outdoor conditions where 'genotype × environment' interactions can mask laboratory-observed responses. Arabidopsis thaliana knockout mutant, uvr8-7, and the corresponding Wassilewskija wild type, were sown outdoors on the same date at 21 locations across Europe, ranging from 39°N to 67°N latitude. Growth and climatic data were monitored until bolting. At the onset of bolting, rosette size, dry weight, and phenolics and glucosinolates were quantified. The uvr8-7 mutant developed a larger rosette and contained less kaempferol glycosides, quercetin glycosides and hydroxycinnamic acid derivatives than the wild type across all locations, demonstrating a role for UVR8 under field conditions. UV effects on rosette size and kaempferol glycoside content were UVR8 dependent, but independent of latitude. In contrast, differences between wild type and uvr8-7 in total quercetin glycosides, and the quercetin-to-kaempferol ratio decreased with increasing latitude, that is, a more variable UV response. Thus, the large-scale synchronized approach applied demonstrates a location-dependent functional role of UVR8 under natural conditions.

The aqueous extract of Cynara cardunculus flowers is traditionally used in cheese production across Mediterranean countries. To meet the growing industrial demand for plant-based milk-clotting enzymes and to explore potential biotechnological applications, we initiated a study to heterologously produce cyprosin B (CYPB), a key milk-clotting enzyme from C. cardunculus, in Nicotiana benthamiana. We also investigated the role of its plant-specific insert (PSI) domain in the CYPB’s activity and its localization. In this study, full-length CYPB and a PSI domain deleted CYPB (CYPBΔPSI) were transiently expressed in N. benthamiana leaves using Agrobacterium-mediated infiltration. The leaves were harvested nine days post-infiltration, and proteins were purified, yielding approximately 81 mg/kg (CYPB) and 60 mg/kg (CYPBΔPSI) fresh weight. CYPBΔPSI showed significantly higher proteolytic activity (156.72 IU/mg) than CYPB (57.2 IU/mg), indicating that the PSI domain is not essential for enzymatic activity and that its removal results in enhanced enzymatic efficiency. In the milk-clotting activity assay, CYPBΔPSI demonstrated a significantly faster clotting time than full-length CYPB, indicating enhanced milk-clotting efficiency for CYPBΔPSI. Subcellular localization studies revealed that CYPB and PSI were localized in the vacuole and endocytic vesicles. In contrast, CYPBΔPSI was primarily localized in the endoplasmic reticulum (ER) and the tonoplast, suggesting that the PSI domain is critical for vacuolar targeting and membrane permeabilization that affects overall protein yield. This study demonstrates the feasibility of using N. benthamiana as a platform for the scalable production of more efficient recombinant CYPB. It highlights the multifunctional role of the PSI domain in vacuolar sorting without impairing its functionality. These results underscore the potential of plant-based expression systems as a viable alternative for the industrial production of plant milk-clotting enzymes, with significant implications for sustainable cheese production.

Phenolics are important secondary metabolites and antioxidants in plants. Mango fruit accumulate abundant phenolic compounds, while the effect of light on the accumulation of different phenolic components in mango peel and the relevant molecular mechanism are still unknown. In this study, mature ‘Guifei’ mango fruit was subjected to postharvest UV-B/white light treatment, fruit peel was sampled for metabolomic and transcriptomic analyses. The results showed that light induced the accumulation of anthocyanins, flavonoids, and phenolics, thus increasing the antioxidant capacity in mango peel. A total of 1,223 phenolic metabolites were detected in mango peel, 36 phenolic compounds were defined as key differentially accumulated metabolites (DAMs) regulated by light. Among the DAMs, the accumulation of 33 compounds was promoted by light, and 30 of these were flavonoids. Light up-regulated most phenolics biosynthesis and light signaling pathway genes, and also regulated expression of plant hormone signaling pathway genes. Transcription factors (TFs) such as MYB, C2H2 and HSF were identified as candidates regulating phenolics biosynthesis in mango. Our findings not only provide information for commercial application of light in promoting mango fruit appearance and increasing phenolics content and antioxidant capacity, but also reveal important aspects of the molecular regulation of light-induced phenolics accumulation in mango peel.

Fruit peel color is an important index of mango fruit quality. Therefore, increasing the anthocyanin accumulation and improving coloration in red mango are crucial for mango industry. The anthocyanin accumulation in mango is light-regulated. However, the effect of white light combined with different doses of UV-B on anthocyanin biosynthesis has not been clarified. Also lacking is a comprehensive analysis of responses of mango fruit peel to UV-B/white light treatments. In this study, green mature ‘Guifei’ mango fruits were subjected to white light combined with low (WL+UV-BL) or high dose UV-B (WL+UV-BH). Anthocyanin concentration, anthocyanin-related gene expression, reactive oxygen species (ROS), antioxidant, and plant hormone concentrations, and antioxidant enzyme activity were measured. The results showed that especially a WL+UV-BH regimen promoted anthocyanin formation in mango peel. Anthocyaninand light signal-related gene expression, ROS content, antioxidant enzyme activity, antioxidant concentrations, and total antioxidant capacity were also increased by UVB/ white light. Such treatments led to higher concentrations of jasmonic acid and cytokines, but decreased content of 1-aminocyclopropane-1-carboxylic acid and salicylic acids. Commercially, our findings may contribute to improving the commercial quality of mango. Scientifically, the present data sheds light on the mango fruit peelspecific molecular and physiological response network under UV-B/white light treatments.

Hydrogen peroxide (H₂O₂) is an important molecule that regulates antioxidant responses that are crucial for plant stress resistance. Exposure to low levels of ultraviolet-B radiation (UV-B, 280–315 nm) can also activate antioxidant defenses and acclimation responses. However, how H₂O₂ and UV-B interact to promote stress acclimation remains poorly understood. In this work, a transgenic model of Nicotiana tabacum cv Xanthi nc, with elevated Mn-superoxide dismutase (Mn-SOD)activity, was used to study the interaction between the constitutive overproduction of H₂O₂ and a 14-day UV-B treatment (1.75 kJ m⁻² d⁻¹ biologically effective UV-B). Subsequently, these plants were subjected to a 7-day moderate drought treatment to evaluate the impact on drought resistance of H₂O₂- and UV-dependent stimulation of the plants' antioxidant system. The UV-B treatment enhanced H₂O₂ levels and altered the antioxidant status by increasing the epidermal flavonol index, Trolox Equivalent Antioxidant Capacity, and catalase, peroxidase and phenylalanine ammonia lyase activities in the leaves. UV-B also retarded growth and suppressed acclimation responses in highly H₂O₂-overproducing transgenic plants. Plants not exposed to UV-B had a higher drought resistance in the form of higher relative water content of leaves. Our data associate the interaction between Mn-SOD transgene overexpression and the UV-B treatment with a stress response. Finally, we propose a hormetic biphasic drought resistance response curve as a function of leaf H₂O₂ content in N. tabacum cv Xanthi.

While the "farm to fork" strategy ticks many boxes in the sustainability agenda, it does not go far enough in addressing how we can improve crop nutraceutical quality. Here, we explored whether supplementary ultraviolet (UV) radiation exposure during growth of broccoli and Chinese cabbage can induce bioactive tryptophan- and glucosinolate-specific metabolite accumulation thereby enhancing Aryl hydrocarbon receptor (AhR) activation in human intestinal cells. By combining metabolomics analysis of both plant extracts and in vitro human colonic fermentation extracts with AhR reporter cell assay, we reveal that human colonic fermentation of UVB-exposed Chinese cabbage led to enhanced AhR activation in human intestinal cells by 23% compared to plants grown without supplementary UV. Thus, by exploring aspects beyond "from farm to fork", our study highlights a new strategy to enhance nutraceutical quality of Brassicaceae, while also providing new insights into the effects of cruciferous vegetables on human intestinal health.

UV-A- or UV-B-enriched growth light were given to basil plants at non-stress-inducing intensities. UV-A-enriched growth light gave rise to a sharp rise in expression of PAL and CHS genes in leaves, an effect that rapidly declined after 1-2 days of exposure. On the other hand, leaves of plants grown in UV-B-enriched light had a more stable and long-lasting increase in expression of these genes and also showed a stronger increase in leaf epidermal flavonol content. UV supplementation of growth light also led to shorter more compact plants with a stronger UV effect the younger the tissue. The effect was more prominent in plants grown under UV-B-enriched light than in those grown under UV-A. Parameters particularly affected were internode lengths, petiole lengths and stem stiffness. In fact, the bending angle of the 2nd internode was found to increase as much as 67% and 162% for plants grown in the UV-A- and UV-B-enriched treatments, respectively. The decreased stem stiffness was probably caused by both an observed smaller internode diameter and a lower specific stem weight, as well as a possible decline in lignin biosynthesis due to competition for precursor by the increased flavonoid biosynthesis. Overall, at the intensities used, UV-B wavelengths are stronger regulators of morphology, gene expression and flavonoid biosynthesis than UV-A wavelengths.

The development of light emitting diodes (LED) gives new possibilities to use the light spectrum to manipulate plant morphology and physiology in plant production and research. Here, vegetative Chrysanthemum × morifolium were grown at a photosynthetic photon flux density of 230 μmol m−2 s−1 under monochromatic blue, cyan, green, and red, and polychromatic red:blue or white light with the objective to investigate the effect on plant morphology, gas exchange and metabolic profile. After 33 days of growth, branching and leaf number increased from blue to red light, while area per leaf, leaf weight fraction, flavonol index, and stomatal density and conductance decreased, while dry matter production was mostly unaffected. Plants grown under red light had decreased photosynthesis performance compared with blue or white light-grown plants. The primary and secondary metabolites, such as organic acids, amino acids and phenylpropanoids (measured by non-targeted metabolomics of polar metabolites), were regulated differently under the different light qualities. Specifically, the levels of reduced ascorbic acid and its oxidation products, and the total ascorbate pool, were significantly different between blue light-grown plants and plants grown under white or red:blue light, which imply photosynthesis-driven alterations in oxidative pressure under different light regimens. The overall differences in plant phenotype, inflicted by blue, red:blue or red light, are probably due to a shift in balance between regulatory pathways controlled by blue light receptors and/or phytochrome. Although morphology, physiology, and metabolism differed substantially between plants grown under different qualities of light, these changes had limited effects on biomass accumulation.

Ultraviolet radiation (UV, 280-400 nm) as an environmental signal triggers metabolic acclimatory responses. However, how different light qualities affect UV acclimation during growth is poorly understood. Here, cucumber plants (Cucumis sativus) were grown under blue, green, red, or white light in combination with UV. Their effects on leaf metabolites were determined using untargeted metabolomics. Blue and white growth light triggered the accumulation of compounds related to primary and secondary metabolism, including amino acids, phenolics, hormones, and compounds related to sugar metabolism and the TCA cycle. In contrast, supplementary UV in a blue or white light background decreased leaf content of amino acids, phenolics, sugars, and TCA-related compounds, without affecting abscisic acid, auxin, zeatin, or jasmonic acid levels. However, in plants grown under green light, UV-induced accumulation of phenolics, hormones (auxin, zeatin, dihydrozeatin-7-N-dihydrozeatin, jasmonic acid), amino acids, sugars, and TCA cycle-related compounds. Plants grown under red light with UV mainly showed decreased sugar content. These findings highlight the importance of the blue light component for metabolite accumulation. Also, data on interactions of UV with green light on the one hand, and blue or white light on the other, further contributes to our understanding of light quality regulation of plant metabolism.