Biology subjects

Strid, A.

Publications and source records attributed to Strid, A..

5 recordsLinked to original sources

bioRxiv2024

Heterologous Production of Cyprosin B in Nicotiana benthamiana: Unveiling the Role of the Plant-Specific Insert Domain in Protein Function and Subcellular Localization

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 CYPBs activity and its localization. In this study, full-length CYPB and a PSI domain deleted CYPB (CYPB{Delta}PSI) 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 (CYPB{Delta}PSI) fresh weight. CYPB{Delta}PSI 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, CYPB{Delta}PSI demonstrated a significantly faster clotting time than full-length CYPB, indicating enhanced milk-clotting efficiency for CYPB{Delta}PSI. Subcellular localization studies revealed that CYPB and PSI were localized in the vacuole and endocytic vesicles. In contrast, CYPB{Delta}PSI 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.

molecular biology↗

bioRxiv2021

UV-B exposure and exogenous hydrogen peroxide application leads to cross-tolerance toward drought in Nicotiana tabacum L.

Acclimation of plants to water deficit involves biochemical and physiological adjustments. Here, we studied how UV-B exposure and exogenously applied hydrogen peroxide (H2O2) potentiates drought tolerance in tobacco (Nicotiana tabacum L.). Separate and combined applications for 14 days of 1.75 kJ m-2 day-1 UV-B radiation and 0.2 mM H2O2 were assessed. Both factors, individually and combined, resulted in inhibition of growth. Furthermore, the combined treatment led to the most compacted plants. UV-B- and UV-B+H2O2-treated plants increased total antioxidant capacity and foliar epidermal flavonol content. H2O2- and UV-B+H2O2-pre-treated plants showed cross-tolerance to a subsequent 7-day drought treatment. Plant responses to the pre-treatment were notably different: i) H2O2 increased the activity of catalase, phenylalanine ammonia lyase and peroxidase activities, and ii) the combined treatment induced epidermal flavonols which were key to drought tolerance. We report synergistic effects of UV-B and H2O2 on transcription accumulation of UV RESISTANCE LOCUS 8, NAC DOMAIN PROTEIN 13 (NAC13), and BRI1-EMS-SUPPRESSOR 1 (BES1). Our data demonstrate a pre-treatment-dependent response to drought for NAC13, BES1 and CHALCONE SYNTHASE transcript accumulation. This study highlights the potential of combining UV-B and H2O2 to improve drought tolerance which could become a useful tool to reduce water use.

plant biology↗

bioRxiv2021

Downsizing in plants - UV induces pronounced morphological changes in cucumber in the absence of stress

Ultraviolet (UV)-A- or UV-B-enrichment of growth light resulted in a stocky cucumber (Cucumis sativus L.) phenotype exhibiting decreased stem and petiole lengths and leaf area. Effects were larger in plants grown in UV-B- than in UV-A-enriched light. In plants grown in UV-A-enriched light, decreases in stem and petiole lengths were similar independently of tissue age. In the presence of UV-B radiation, stems and petioles were progressively shorter the younger the tissue. Also, plants grown under UV-A-enriched light significantly reallocated photosynthate from shoot to root and also had thicker leaves with decreased specific leaf area. Our data therefore imply different morphological plant regulatory mechanisms under UV-A and UV-B radiation. There was no evidence of stress in the UV-exposed plants, neither in photosynthetic parameters, total chlorophyll content, nor in accumulation of damaged DNA (cyclobutane pyrimidine dimers). The ABA content of the plants also was consistent with non-stress conditions. Parameters such as total leaf antioxidant activity, leaf adaxial epidermal flavonol content and foliar total UV-absorbing pigment levels revealed successful UV acclimation of the plants. Thus, the stocky UV-phenotype was displayed by healthy cucumber plants, implying a strong morphological response and regulatory adjustment as part of UV acclimation processes involving UV-A and/or UV-B photoreceptors.

plant biology↗

bioRxiv2020

Management implications based on diversity patterns under climate change scenarios in a continental island biodiversity hotspot

In the Anthropocene era, climate change poses a great challenge in environmental management and decision-making for species and habitat conservation. To support decision-making, many studies exist regarding the expected vegetation changes and the impacts of climate change on European plants, yet none has investigated how climate change will affect the extinction risk of the entire endemic flora of an island biodiversity hotspot, with intense human disturbance. Our aim is to assess, in an integrated manner, the impact of climate change on the biodiversity and biogeographical patterns of Crete and to provide a case-study upon which a cost-effective and climate-smart conservation planning strategy might be set. We employed a variety of macroecological analyses and estimated the current and future biodiversity, conservation and extinction hotspots in Crete, as well as the factors that may have shaped these distribution patterns. We also evaluated the effectiveness of climate refugia and the NATURA 2000 network (PAs) on protecting the most vulnerable species and identified the taxa that should be of conservation priority based on the Evolutionary Distinct and Globally Endangered (EDGE) index, during any environmental management process. The highlands of Cretan mountain massifs have served as both diversity cradles and museums, due to their stable climate and high topographical heterogeneity. They are also identified as biodiversity hotspots, as well as areas of high conservation and evolutionary value, due their high EDGE scores. Due to the escalator to extinction phenomenon and the subsequent biotic homogenization, these areas are projected to become diversity death-zones in the near future and should thus be prioritized in terms of conservation efforts and by decision makers. In-situ conservation focusing at micro-reserves and ex-situ conservation practices should be considered as an insurance policy against such biodiversity losses, which constitute cost-effective conservation measures. Scientists and authorities should aim the conservation effort at areas with overlaps among PAs and climate refugia, characterized by high diversity and EDGE scores. These areas may constitute Anthropocene refugia. Thus, this climate-smart, cost-effective conservation-prioritization planning will allow the preservation of evolutionary heritage, trait diversity and future services for human well-being and acts as a pilot for similar regions worldwide.

ecology↗

bioRxiv2020

The photoreceptor UVR8 mediates the perception of both UV-B and UV-A wavelengths up to 350 nm of sunlight with responsivity moderated by cryptochromes

The photoreceptors UV RESISTANCE LOCUS 8 (UVR8) and CRYPTOCHROMES 1 and 2 (CRYs) play major roles in the perception of UV-B (280-315 nm) and UV-A/blue radiation (315-500 nm), respectively. However, it is poorly understood how they function in sunlight. The roles of UVR8 and CRYs were assessed in a factorial experiment with Arabidopsis thaliana wild-type and photoreceptor mutants exposed to sunlight for 6 h or 12 h under five types of filters with cut-offs in UV and blue-light regions. Transcriptome-wide responses triggered by UV-B and UV-A wavelengths shorter than 350 nm (UV-Asw) required UVR8 whereas those induced by blue and UV-A wavelengths longer than 350 nm (UV-Alw) required CRYs. UVR8 modulated gene expression in response to blue light while lack of CRYs drastically enhanced gene expression in response to UV-B and UV-Asw. These results agree with our estimates of photons absorbed by these photoreceptors in sunlight and with in vitro monomerization of UVR8 by wavelengths up to 335 nm. Motif enrichment analysis predicted complex signaling downstream of UVR8 and CRYs. Our results highlight that it is important to use UV waveband definitions specific to plants photomorphogenesis as is routinely done in the visible region.

plant biology↗