The labile zinc pool in plant cells Zlobin, Ilya E; Kartashov, Alexander V; Nosov, Alexander V ...
Functional plant biology : FPB,
01/2019, Letnik:
46, Številka:
9
Journal Article
Recenzirano
Zinc is the most abundant and important transition metal in plants; however, the dynamic aspects of zinc homeostasis in plant cells are poorly understood. In this study we explored the pool of labile ...exchangeable zinc complexes in plant cells, and the potential influence of changes in intracellular zinc availability on cellular physiology. Work was performed on cultivated cell extracts of Arabidopsis thaliana (L.) Heynh. and Thellungiella salsuginea (Pall.) O.E. Schulz grown under control (3.48 µM Zn2+), 10-fold Zn excess or Zn starvation conditions. The free and labile Zn contents in the extracts were then determined by fluorimetric titration. We observed for the first time that plant cells contain micromolar concentrations of labile zinc complexes that account for a low percentage of the total zinc content. Labile zinc is mainly protein bound. Zn starvation inhibits cell proliferation and leads to the disappearance of the labile zinc pool, whereas Zn excess drastically increases the labile zinc pool. Free Zn2+ is buffered at picomolar concentrations in the intracellular milieu, and the increase in free Zn2+ concentrations to low nanomolar values clearly modulates enzyme activity by direct reversible binding. Such increases in free Zn2+ can be achieved by the substantial influx of additional zinc or by the oxidation of zinc-binding thiols. The observed features of the labile zinc pool in plant cells suggest it has a role in intracellular zinc trafficking and zinc signalling.
Callus and suspension cell cultures were successfully developed from
Sutherlandia frutescens
(Fabaceae), an endemic medicinal plant of South Africa. Two callus cell lines, originating from hypocotyl ...and cotyledon explants of in vitro seedlings under both dark and light conditions, showed intensive fresh weight accumulation with growth index ranging from 4.6 to 5.9. Suspension cell cultures induced from two callus lines had similar growth profiles and their growth index (15–18), specific growth rate (0.15–0.16 day
−1
), productivity (0.83–0.96 g/(l day)) and maximum biomass accumulation (16–18 g/l) remained relatively high for Fabaceae cell cultures during 27 sub-cultivations. Callus and suspension cell cultures showed similar profiles of secondary metabolites that were, however, different from leaves of greenhouse plants. Isoflavones were predominant in both callus and suspension cell cultures while flavonoids (sutherlandins) and triterpene glycosides of the cycloartane group (sutherlandiosides) were mostly found in leaves. Nineteen fatty acids (FA), both short- and very-long-chained (up to C
25:0
), were found in cell cultures. Linoleic and α-linolenic FA together comprised 60–64% out of total FA content in cell cultures followed by palmitic acid (18–25%). Extracts of suspension cell biomass exhibited antimicrobial activity against
Staphylococcus aureus
but were not effective against
Pseudomonas aeruginosa.
To the best of our knowledge, this is the first report on the induction, phytochemical composition and antimicrobial activity screening of
S. frutescens
suspension cell cultures which opens the door for their biotechnological application.
The involvement of endocytosis in Na⁺ intenalization by suspension-cultured Arabidopsis thaliana (L.) Heynh. cells under salt stress was investigated. Using epifluorescence and confocal ...laser-scanning microscopy in co-localization experiments with a marker of the endocytic structures, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide (FM 4-64), and a membrane-impermeable Na⁺ indicator, Asante Natrium Green-2 tetramethylammonium salt (ANG-2 TMA), intracellular Na⁺ ion uptake via endocytosis, but not through the plasma-membrane ion channels or transporters, was detected. The punctate fluorescence of the two markers was co-localized in the microvacuoles (MVs) with sizes up to 5 µm. The study of cell ultrastructure by transmission electron microscopy revealed the fusion of MVs into larger ones during an incubation of the cells in medium containing 100 mM NaCl. Wortmannin, an inhibitor of multivesicular body/late endosome/prevacuolar compartment fusion with vacuoles, substantially lowered intracellular Na⁺ content. Apparently, endocytic Na⁺ uptake and subsequent fusion of Na⁺-containing membrane structures provided direct transport of external Na⁺ ions into the vacuoles, bypassing the cytosol.
Ethylene is known to influence the cell cycle (CC) via poorly characterized roles whilst nitric oxide (NO) has well-established roles in the animal CC but analogous role(s) have not been reported for ...plants. As NO and ethylene signaling events often interact we examined their role in CC in cultured cells derived from
wild-type (Col-0) plants and from ethylene-insensitive mutant
plants. Both NO and ethylene were produced mainly during the first 5 days of the sub-cultivation period corresponding to the period of active cell division. However, in
cells, ethylene generation was significantly reduced while NO levels were increased. With application of a range of concentrations of the NO donor, sodium nitroprusside (SNP) (between 20 and 500 μM) ethylene production was significantly diminished in Col-0 but unchanged in
cells. Flow cytometry assays showed that in Col-0 cells treatments with 5 and 10 μM SNP concentrations led to an increase in S-phase cell number indicating the stimulation of G1/S transition. However, at ≥20 μM SNP CC progression was restrained at G1/S transition. In the mutant
strain, the index of S-phase cells was not altered at 5-10 μM SNP but decreased dramatically at higher SNP concentrations. Concomitantly, 5 μM SNP induced transcription of genes encoding
and
in Col-0 cells whereas transcription of
s and
s were not significantly altered in
cells at any SNP concentrations examined. Hence, it is appears that EIN2 is required for full responses at each SNP concentration. In
cells, greater amounts of NO, reactive oxygen species, and the tyrosine-nitrating peroxynitrite radical were detected, possibly indicating NO-dependent post-translational protein modifications which could stop CC. Thus, we suggest that in
cultured cells NO affects CC progression as a concentration-dependent modulator with a dependency on EIN2 for both ethylene production and a NO/ethylene regulatory function.
•Free Zn2+ levels regulate the expression of Zn homeostasis genes in Arabidopsis cells.•Zn deficiency influences protein folding in the cytosol of Arabidopsis cells.•mRNA degradation of Zn ...homeostasis genes is influenced by free Zn2+ levels.
Zinc is one of the most widespread transition metals in plants, and Zn deficiency has many adverse effects on plant productivity and human health. The zinc homeostasis system is actively studied; however, the mechanisms underlying regulation of Zn homeostasis have not been fully elucidated. Two potential ways to assess cellular Zn homeostasis are to sense changes in the free Zn2+ levels or disturbances in protein folding due to zinc deficiency. We found that changes in the cellular free Zn2+ level regulated the transcriptional activity of a set of primary Zn homeostasis genes in a dose-dependent manner and with fast kinetics. We propose that in addition to the well-known bZIP19/bZIP23 transcription factors, another Zn-sensing system exists in the cell and regulates free Zn2+-dependent changes in MTP2 gene expression. We first demonstrated that changes in the free Zn2+ levels in the cells likely influenced not only the transcriptional activation but also the mRNA degradation rates of Zn homeostasis genes, which were accelerated for several genes during recovery of the cellular Zn status. Zn deficiency clearly disturbed the protein folding processes in the cytosol of plant cells, but the cellular response system to these disturbances did not play a significant role in the regulation of Zn homeostasis genes.
An environmentally friendly, resource-efficient substitute for the production of vegetative biomass and the bioactive components of medicinal plants is provided by novel biotechnology-based ...suspension cells cultured in bioreactors. Despite the cultured biomass is produced in the controlled conditions, it requires detailed chemical analysis and toxicological evaluation before being approved for human consumption. This study reports the results of biochemical, toxicological, and elemental composition analysis of the suspension cell culture of a medicinal species Panax japonicus (T. Nees) C.A. Mey. cultivated in a large-scale bioreactor system. For the scaling up the process of cultivation, the suspension culture was grown sequentially in a series of laboratory (20-L), pilot (75-L), and industrial (630-L) bioreactors. The biomass productivity of cell culture was comparable or higher than of wild plants or in vitro cultured hairy roots reported in the literature. The resulting cell biomass was analyzed for ginsenoside contents using ultra-high-performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC–ESI–MS). For the elemental composition of cells, inductively coupled argon plasma mass spectrometry (ICP–MS) was used. In addition, the biomass was evaluated for acute toxicity and skin sensibilization effects on in vivo animal models. The analyses confirmed that suspension cells cultured in bioreactors accumulate a broad spectrum of ginsenosides and their malonylated derivatives at a total concentration of 7.54 % (w/w) based on dry weight and contain essential macro- (K, Ca, Mg, Na) and micro- (Zn, Mn, Fe, B, Al, Cu) elements in dietary-safe concentrations. Acute toxicity tests using the administered doses of 2000 and 5000 mg dry biomass per kg animal weight resulted in no changes in animal organ weights and no or minor changes in hematological and biochemical parameters of blood. No skin irritation or sensitizing effects were observed. Therefore, bioreactor cultivation of P. japonicus cell suspension is highly productive and provides a ginsenoside-rich and non-toxic cell biomass which is a potentially valuable component of functional foods, food additives, and natural health products.
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•Panax japonicus is a medicinal ginseng used in functional foods and health products.•Cell culture is a sustainable approach for biomass and ginsenoside production.•Panax japonicus cell culture was produced in bioreactors of 20, 75 and 630 L.•Cell biomass contained essential macro- and microelements and 7.53 % of ginsenosides.•Cell biomass showed no toxicity on rats based on blood parameters and organ weight.
The article deals with establishment and development of Russian Nationalist Movement in megalopolis on the verge of 1980’s – 1990’s. The key features of the period are uncovered in this article
Ex situ collections of algae, cyanobacteria, and plant materials (cell cultures, hairy and adventitious root cultures, shoots, etc.) maintained in vitro or in liquid nitrogen (-196 °C, LN) are ...valuable sources of strains with unique ecological and biotechnological traits. Such collections play a vital role in bioresource conservation, science, and industry development but are rarely covered in publications. Here, we provide an overview of five genetic collections maintained at the Institute of Plant Physiology of the Russian Academy of Sciences (IPPRAS) since the 1950-1970s using in vitro and cryopreservation approaches. These collections represent different levels of plant organization, from individual cells (cell culture collection) to organs (hairy and adventitious root cultures, shoot apices) to in vitro plants. The total collection holdings comprise more than 430 strains of algae and cyanobacteria, over 200 potato clones, 117 cell cultures, and 50 strains of hairy and adventitious root cultures of medicinal and model plant species. The IPPRAS plant cryobank preserves in LN over 1000 specimens of in vitro cultures and seeds of wild and cultivated plants belonging to 457 species and 74 families. Several algae and plant cell culture strains have been adapted for cultivation in bioreactors from laboratory (5-20-L) to pilot (75-L) to semi-industrial (150-630-L) scale for the production of biomass with high nutritive or pharmacological value. Some of the strains with proven biological activities are currently used to produce cosmetics and food supplements. Here, we provide an overview of the current collections' composition and major activities, their use in research, biotechnology, and commercial application. We also highlight the most interesting studies performed with collection strains and discuss strategies for the collections' future development and exploitation in view of current trends in biotechnology and genetic resources conservation.
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