Chloroplast Sec14-like 1 (CPSFL1) is essential for normal chloroplast development and affects carotenoid accumulation inChlamydomonas

Plastid isoprenoid-derived carotenoids serve essential roles in chloroplast development and photosynthesis. Although nearly all enzymes that participate in the biosynthesis of carotenoids in plants have been identified, the complement of auxiliary proteins that regulate synthesis, transport, sequestration, and degradation of these molecules and their isoprenoid precursors have not been fully described. To identify such proteins that are necessary for the optimal functioning of oxygenic photosynthesis, we screened a large collection of nonphotosynthetic (acetate-requiring) DNA insertional mutants ofChlamydomonas reinhardtiiand isolatedcpsfl1. Thecpsfl1mutant is extremely light-sensitive and susceptible to photoinhibition and photobleaching. TheCPSFL1gene encodes a CRAL-TRIO hydrophobic ligand-binding (Sec14) domain protein. Proteins containing this domain are limited to eukaryotes, but some may have been retargeted to function in organelles of endosymbiotic origin. Thecpsfl1mutant showed decreased accumulation of plastidial isoprenoid-derived pigments, especially carotenoids, and whole-cell focused ion-beam scanning-electron microscopy revealed a deficiency of carotenoid-rich chloroplast structures (e.g., eyespot and plastoglobules). The low carotenoid content resulted from impaired biosynthesis at a step prior to phytoene, the committed precursor to carotenoids. The CPSFL1 protein bound phytoene and β-carotene when expressed inEscherichia coliand phosphatidic acid in vitro. We suggest that CPSFL1 is involved in the regulation of phytoene synthesis and carotenoid transport and thereby modulates carotenoid accumulation in the chloroplast.

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The structure of the COPI coat determined within the cell

eLife ◽

10.7554/elife.32493 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 60

Author(s):

Yury S Bykov ◽

Miroslava Schaffer ◽

Svetlana O Dodonova ◽

Sahradha Albert ◽

Jürgen M Plitzko ◽

...

Keyword(s):

Focused Ion Beam ◽

Structural Model ◽

Electron Tomography ◽

Ion Beam ◽

Membrane Thickness ◽

In Vitro Reconstitution ◽

Cryo Electron Tomography ◽

Subtomogram Averaging

COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. Previously we have determined a complete structural model of the in vitro reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath β’–COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant.

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Herbicides which Inhibit Electron Transport or Produce Chlorosis and Their Effect on Chloroplast Development in Radish Seedlings. III. Plastid Pigment and Quinone Composition

Zeitschrift für Naturforschung C ◽

10.1515/znc-1982-7-813 ◽

1982 ◽

Vol 37 (7-8) ◽

pp. 642-650 ◽

Cited By ~ 3

Author(s):

K. H. Grumbach

Keyword(s):

Light Quality ◽

Chloroplast Development ◽

Red Light ◽

Carotenoid Content ◽

Bleaching Effect ◽

Low Light ◽

Chloroplast Pigments ◽

Light Intensities ◽

Radish Seedlings ◽

Β Carotene

Abstract The effect of DCMU, bentazon, amitrole and SAN 6706 on the formation of chloroplast pigments and quinones was investigated using plants that were grown in total darkness or continuous white, red or far-red light. All herbicides assayed affected the formation of chlorophylls, carotenoids and quinones but DCMU had only minor effects. Like for chlorophylls and carotenoids the formation of quinones was most suppressed in plants grown in the presence of the herbicide in continuous white or red light, but the effect on the formation of quinones was much lower as compared to the pigments. The observation that the biosynthesis of quinones is still maintained in SAN 6706 treated bleached plastids which are lacking chlorophylls and carotenoids indicates that quinones are synthesized at the plastitd envelope and stored in the osmiophilic plastoglobuli. Amitrole and SAN 6706 induced a strong chlorosis. It was of particular interest that chlorosis was also induced by the photosystem II inhibitor bentazon. DCMU was not effective. The inhibitor concentration for 50% inhibition in the chlorophyll and carotenoid content was 5 × 10-6 ᴍ for SAN 6706,3 × 10-4 ᴍ for amitrole and 10-3 ᴍ for bentazon. As already reported by others SAN 6706 treated plants accumulated phytoene in large amounts. The highest phytoene content was observed in plants that were grown in the dark. Amitrole treated plants accumulated lycopene. But in addition other carotenoid precursors like phytoene and phytofluene were also accumulated. In contrast to phytoene lycopene was only accumulated in plants that were grown in the light. Particularly for SAN 6706 and amitrole the expression of the bleaching effect was depending on the light intensity and light quality that was used during plant growth. The herbicide effect ewas predominantly expressed at higher light intensities and after irradiation with red light. The observation that the induction of chlorosis is very sensitive to red light as compared to white or blue light is suggesting that phytochrome is involved in the development of the herbicide toxicity. It also supports that in SAN treated plants chlorophylls are photodecomposed directly by light because of the lack of photoprotecting carotenoids but mainly β-carotene in these plastids. Further support for this was given by the demonstration that SAN treated plants which were grown at very low light intensities turned green and were photosynthetically active.

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Enhanced Cells Anchoring to Electrospun Hybrid Scaffolds With PHBV and HA Particles for Bone Tissue Regeneration

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.632029 ◽

2021 ◽

Vol 9 ◽

Author(s):

Joanna E. Karbowniczek ◽

Łukasz Kaniuk ◽

Krzysztof Berniak ◽

Adam Gruszczyński ◽

Urszula Stachewicz

Keyword(s):

Cell Proliferation ◽

Bone Tissue ◽

Tissue Regeneration ◽

Focused Ion Beam ◽

Ion Beam ◽

Inorganic Compounds ◽

Bone Tissue Regeneration ◽

Sem Imaging ◽

Hybrid Scaffolds

Hybrid materials combining organic and inorganic compounds used as scaffolds are highly beneficial in bone regeneration. In this study, we successfully produced by blend electrospinning poly(3-hydroxybutyric acid-co-3-hydrovaleric acid) (PHBV) scaffolds enriched with hydroxyapatite (HA) particles to biomimic bone tissue for improved and faster regeneration processes. The morphology, fiber diameters, and composition of the scaffolds were investigated by scanning electron microscopy (SEM) techniques followed by focused ion beam (FIB) sectioning to verify HA particles integration with PHBV fibers. In vitro cell culture was performed for 7 days and followed with the cell proliferation test (CellTiter-Blue® Assay). Additionally, cell integration with the scaffold was visualized by confocal and SEM imaging. We developed a simple way of obtaining hybrid scaffolds by electrospinning PHBV solution with HA particles without any post-processing. The PHBV + HA scaffold enhanced cell proliferation and filopodia formation responsible for cell anchoring within the created 3D environment. The obtained results show the great potential in the development of hybrid scaffolds stimulating bone tissue regeneration.

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In vitro Evaluation of Structural Factors Favouring Bacterial Adhesion on Orthodontic Adhesive Resins

10.20944/preprints202103.0322.v1 ◽

2021 ◽

Author(s):

Roberta Condò ◽

Gianluca Mampieri ◽

Guido Pasquantonio ◽

Aldo Giancotti ◽

Paola Pirelli ◽

...

Keyword(s):

Surface Roughness ◽

Bacterial Adhesion ◽

Focused Ion Beam ◽

Ion Beam ◽

Structural Factors ◽

Adhesive Material ◽

Internal Structures ◽

Structural Nature ◽

Beam Technique

Bacterial adhesion to the surface of the adhesive material is an important step in the formation of plaque and enamel demineralization. In order to correlate the material composition to the specific surface roughness of the resin and to the probable more favourable adhesion of bacteria, scanning electron microscopy, combined with focus ion bean micromachining, together with stylus profilometry analysis have been in vitro performed to reveal the structural nature of three orthodontic adhesive resins used for bracket bonding and, above all, to understand how compositional factors can influence specific pivotal properties such as material’s surface roughness and robustness. In particular, we speculated about the morphological features that determine an increase in the bacterial adhesion and we proposed focused ion beam technique as a valuable tool to compare the internal structures of the polymers and to determine the peculiar mechanical properties of the examined adhesive resins.

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Effects of Exogenous Abscisic Acid (ABA) on Carotenoids and Petal Color in Osmanthus fragrans ‘Yanhonggui’

Plants ◽

10.3390/plants9040454 ◽

2020 ◽

Vol 9 (4) ◽

pp. 454

Author(s):

Yucheng Liu ◽

Bin Dong ◽

Chao Zhang ◽

Liyuan Yang ◽

Yiguang Wang ◽

...

Keyword(s):

Abscisic Acid ◽

Metabolic Pathway ◽

Carotenoid Biosynthesis ◽

Carotenoid Content ◽

Native Plant ◽

Total Carotenoids ◽

Osmanthus Fragrans ◽

Carotenoid Accumulation ◽

Β Carotene ◽

Carotenoid Degradation

Osmanthus fragrans is a well-known native plant in China, and carotenoids are the main group of pigments in the petals. Abscisic acid (ABA) is one of the products of the metabolic pathway of carotenoids. Application of ABA could affect pigmentation of flower petals by changing the carotenoid content. However, little is known about the effects of ABA treatment on carotenoid accumulation in O. fragrans. In this study, different concentrations of ABA (0, 150 and 200 mg/L) were spread on the petals of O. fragrans ‘Yanhonggui’. The petal color of ‘Yanhonggui’ receiving every ABA treatment was deeper than that of the control. The content of total carotenoids in the petals significantly increased with 200 mg/L ABA treatment. In the petals, α-carotene and β-carotene were the predominant carotenoids. The expression of several genes involved in the metabolism of carotenoids increased with 200 mg/L ABA treatment, including PSY1, PDS1, Z-ISO1, ZDS1, CRTISO, NCED3 and CCD4. However, the transcription levels of the latter two carotenoid degradation-related genes were much lower than of the five former carotenoid biosynthesis-related genes; the finding would explain the significant increase in total carotenoids in ‘Yanhonggui’ petals receiving the 200 mg/L ABA treatment.

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HPLC-UV/Vis-APCI-MS/MS Determination of Major Carotenoids and Their Bioaccessibility from “Delica” (Cucurbita maxima) and “Violina” (Cucurbita moschata) Pumpkins as Food Traceability Markers

Molecules ◽

10.3390/molecules23112791 ◽

2018 ◽

Vol 23 (11) ◽

pp. 2791 ◽

Cited By ~ 5

Author(s):

Caterina Bergantin ◽

Annalisa Maietti ◽

Paola Tedeschi ◽

Guillermina Font ◽

Lara Manyes ◽

...

Keyword(s):

Food Processing ◽

Carotenoid Content ◽

Cooking Methods ◽

Cucurbita Moschata ◽

Cucurbita Maxima ◽

Steam Cooking ◽

In Vitro Bioaccessibility ◽

Β Carotene

Carotenoids are a widespread group of fat-soluble pigments, and their major nutritional importance comes from their pro-vitamin A activity and their antioxidant capacity. In this study, two different pumpkin cultivars (Cucurbita maxima, also named `Delica’ and Cucurbita moschata, also known as `Violina’) from the southern Po Delta area were investigated in terms of carotenoid content and the influence of food processing on compositional changes and carotenoid bioaccessibility. Quali- and quantitative determination of carotenoids in sample extracts were performed on a C30 column by means of an online coupled HPLC-UV/Vis-APCI-MS/MS technique. The identification of separated compounds was tentatively achieved by merging (i) chromatographic data, (ii) UV-Vis spectra, and (iii) MS/MS fragmentation spectra. The chromatographic profiles for the two cultivars showed qualitative differences. Two major carotenoids were considered for quantification purposes and further investigations: lutein and β -carotene. Quantification of target carotenoids was performed with external calibration through analytical standards. The concentration of lutein and β -carotene was higher in C. maxima than in the other variety, C. moschata. Carotenoids are susceptible to degradation (isomerization and oxidation) during food processing (i.e., cooking), and the concentration of lutein and β -carotene were monitored in oven-cooked and steam-cooked pumpkins. The steam-cooking process was superior in terms of limiting carotenoid loss. A complete functional profile of pumpkins as a source of carotenoids was gained with the evaluation of their in vitro bioaccessibility and their bioavailability after intake during human digestion. Bioaccessibility of lutein and β -carotene were estimated by an in vitro static digestion model that involved salivary, gastric, and duodenal phases. Bioaccessibility values progressively increased from the salivary to the duodenal phase for both pumpkin varieties and cooking methods. Bioaccessibility of lutein was always lower than β -carotene for both cultivars and for both cooking methods. Bioaccessibility values for lutein and β -carotene changed from 1.93% to 2.34% vs. 4.94% and 8.83% in the salivary phase, from 2.7% to 4.63% vs. 7.83% and 15.60% in the gastric phase, and from 10.04% to 13.42% vs. 25.81% and 35.32% in the duodenal phase. For both target compounds, bioaccessibility in the duodenal phase was more than twice the gastric values, and it underlined that the type of cooking did not influence release from the initial matrix.

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Repression of Carotenoid Accumulation by Nitrogen and NH4+ Supply in Carrot Callus Cells In Vitro

Plants ◽

10.3390/plants10091813 ◽

2021 ◽

Vol 10 (9) ◽

pp. 1813

Author(s):

Tomasz Oleszkiewicz ◽

Michał Kruczek ◽

Rafal Baranski

Keyword(s):

Callus Growth ◽

Carotenoid Content ◽

N Availability ◽

Carotenoid Accumulation ◽

Carotenoid Metabolism ◽

Ammonium N ◽

Dark Orange ◽

Modified Media ◽

Orange Color

The effect of mineral nutrition on the accumulation of the main health beneficial compounds in carrots, the carotenoid pigments, remains ambiguous; here, a model-based approach was applied to reveal which compounds are responsible for the variation in carotenoid content in carrot cells in vitro. For this purpose, carotenoid-rich callus was cultured on either BI (modified Gamborg B5) or R (modified Murashige and Skoog MS) mineral media or on modified media obtained by exchanging compounds between BI and R. Callus growing on the BI medium had abundant carotene crystals in the cells and a dark orange color in contrast to pale orange callus with sparse crystals on the R medium. The carotenoid content, determined by HPLC and spectrophotometrically after two months of culture, was 5.3 higher on the BI medium. The replacement of media components revealed that only the N concentration and the NO3:NH4 ratio affected carotenoid accumulation. Either the increase of N amount above 27 mM or decrease of NO3:NH4 ratio below 12 resulted in the repression of carotenoid accumulation. An adverse effect of the increased NH4+ level on callus growth was additionally found. Somatic embryos were formed regardless of the level of N supplied. Changes to other media components, i.e., macroelements other than N, microelements, vitamins, growth regulators, and sucrose had no effect on callus growth and carotenoid accumulation. The results obtained from this model system expand the range of factors, such as N availability, composition of N salts, and ratio of nitrate to ammonium N form, that may affect the regulation of carotenoid metabolism.

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Spray-dried lactose-leucine microparticles for pulmonary delivery of antimycobacterial nanopharmaceuticals

Drug Delivery and Translational Research ◽

10.1007/s13346-021-01011-7 ◽

2021 ◽

Author(s):

Durairaj Thiyagarajan ◽

Benedikt Huck ◽

Birgit Nothdurft ◽

Marcus Koch ◽

David Rudolph ◽

...

Keyword(s):

Spray Drying ◽

Focused Ion Beam ◽

Colloidal Stability ◽

Ion Beam ◽

Pulmonary Delivery ◽

Active Pharmaceutical Ingredient ◽

Dry Powder ◽

Aerodynamic Properties ◽

Model Cell

AbstractPulmonary delivery of nanocarriers for novel antimycobacterial compounds is challenging because the aerodynamic properties of nanomaterials are sub-optimal for such purposes. Here, we report the development of dry powder formulations for nanocarriers containing benzothiazinone 043 (BTZ) or levofloxacin (LVX), respectively. The intricacy is to generate dry powder aerosols with adequate aerodynamic properties while maintaining both nanostructural integrity and compound activity until reaching the deeper lung compartments. Microparticles (MPs) were prepared using vibrating mesh spray drying with lactose and leucine as approved excipients for oral inhalation drug products. MP morphologies and sizes were measured using various biophysical techniques including determination of geometric and aerodynamic mean sizes, X-ray diffraction, and confocal and focused ion beam scanning electron microscopy. Differences in the nanocarriers’ characteristics influenced the MPs’ sizes and shapes, their aerodynamic properties, and, hence, also the fraction available for lung deposition. Spay-dried powders of a BTZ nanosuspension, BTZ-loaded silica nanoparticles (NPs), and LVX-loaded liposomes showed promising respirable fractions, in contrast to zirconyl hydrogen phosphate nanocontainers. While the colloidal stability of silica NPs was improved after spray drying, MPs encapsulating either BTZ nanosuspensions or LVX-loaded liposomes showed the highest respirable fractions and active pharmaceutical ingredient loads. Importantly, for the BTZ nanosuspension, biocompatibility and in vitro uptake by a macrophage model cell line were improved even further after spray drying. Graphical abstract

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Expression of a Chromoplast-Specific Lycopene β-Cyclase Gene (CYC-B) Is Implicated in Carotenoid Accumulation and Coloration in the Loquat

Biomolecules ◽

10.3390/biom9120874 ◽

2019 ◽

Vol 9 (12) ◽

pp. 874 ◽

Cited By ~ 3

Author(s):

Min Hong ◽

Zhuo-Heng Chi ◽

Yong-Qing Wang ◽

Yue-Ming Tang ◽

Qun-Xian Deng ◽

...

Keyword(s):

Gene Expression ◽

Metabolic Pathway ◽

Negative Control ◽

Carotenoid Content ◽

Virus Induced Gene Silencing ◽

Carotenoid Accumulation ◽

Homologous Genes ◽

Total Carotenoid Content ◽

The Difference ◽

Β Carotene

Carotenoids are the principal pigments in the loquat. Although the metabolic pathway of plant carotenoids has been extensively investigated, few studies have been explored the regulatory mechanisms of loquat carotenoids because knowledge of the loquat genome is incomplete. The chromoplast-specific lycopene β-cyclase gene (CYC-B) could catalyze cyclization of lycopene to β-carotene. In this study, the differential accumulation patterns of loquat with different colors were analyzed and virus-induced gene silencing (VIGS) was utilized in order to verify CYC-B gene function. Using a cloning strategy of homologous genes, a CYC-B gene orthologue was successfully identified from the loquat. At a later stage of maturation, CYC-B gene expression and carotenoids concentrations in the ‘Dawuxing’ variety were higher than in ‘Chuannong 1-5-9′, possibly leading to the difference in pulp coloration of loquat. Interference of CYC-B gene expression in the loquat demonstrated clear visual changes. The green color in negative control fruits became yellow, while TRV2-CYC-B silenced fruits remained green. CYC-B gene expression and total carotenoid content in the pulp decreased by 32.5% and 44.1%, respectively. Furthermore, multiple key genes in the carotenoid metabolic pathway synergistically responded to downregulation of CYC-B gene expression. In summary, we provide direct evidences that CYC-B gene is involved in carotenoid accumulation and coloration in the loquat.

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Postharvest UV-B and Photoperiod with Blue + Red LEDs as Strategies to Stimulate Carotenogenesis in Bell Peppers

Applied Sciences ◽

10.3390/app11093736 ◽

2021 ◽

Vol 11 (9) ◽

pp. 3736

Author(s):

Lorena Martínez-Zamora ◽

Noelia Castillejo ◽

Francisco Artés-Hernández

Keyword(s):

Carotenoid Content ◽

Fluorescent Light ◽

Light Conditions ◽

Retail Sale ◽

Carotenoid Accumulation ◽

Bell Peppers ◽

Total Antioxidant ◽

Bioactive Content ◽

Red Leds ◽

Β Carotene

Background: Our objective was to evaluate carotenoid accumulation in bell peppers during shelf life under different light conditions. Methods: Fruit stored for 6 d at 7 °C received a 9 kJ m−2 UV-B treatment, while non-UV-treated were used as control (CTRL). Subsequently, all peppers were disposed for a retail sale period of 4 d at 20 °C with a photoperiod of 14 h under fluorescent light (FL) + 10 h under darkness (D), FL, or blue + red LEDs (BR LED). Results: Total antioxidant capacity (TAC) was increased by the UV-B treatment and the photoperiods supplemented with FL and BR LED, which was directly related to the carotenoid content. In fact, CTRL peppers (225 mg β-carotene kg−1) under FL+BR LED showed an increase of ~33% of 13-cis-β-carotene, ~24% of all-trans-β-carotene, and ~27.5% of 9-cis-β-carotene compared to FL + D and FL + FL. Capsaicinoids showed an increase by ~22%, ~38%, and ~27% in the content of capsanthin, capsanthin laurate, and capsanthin esters, respectively, after the UV-B treatment, which was even enhanced after the LED-supplemented photoperiod by ~18% compared to FL+D. Conclusions: Illumination with BR LEDs + UV-B during the retail sale period nights is recommended to increase the bioactive content of bell peppers via carotenoid accumulation to 270 mg β-carotene kg−1.

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