scholarly journals Characterization of Suspension Cultures of Arabidopsis thaliana (L.) Heynh Cells with Altered Expression of the Gene of Alternative Mitochondrial Oxidase Aox1a and Analysis of their Frost Resistance

2021 ◽  
Vol 35 ◽  
pp. 3-18
Author(s):  
A. V. Stepanov ◽  
◽  
S. A. Kashin ◽  
N. S. Zabanova ◽  
O. A. Fedotova ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Suspension Culture ◽  
Mitochondrial Respiration ◽  
Frost Resistance ◽  
Alternative Oxidase ◽  
Uncoupling Protein ◽  
Living Cells ◽  
Subzero Temperature ◽  
Wild Type ◽  
Culture Cells

The enzyme alternative cyanide-resistant oxidase (AOX) localized in mitochondria is involved in the processes of plant adaptation to various unfavorable biotic and abiotic factors. Transfer of electrons from ubiquinone to oxygen by alternative oxidase has a nonprotonmotive character and, by bypassing two sites of H+ pumping in complexes III and IV, lowers the energy efficiency of respiration and energy of electron flow through AOX is released as heat. In this work, we characterized heterotrophic suspension cultures of Arabidopsis thaliana (L.) Heynh cells obtained from seeds of plants with altered (reduced (AS-12 line) and increased (XX-2 line)) expression of the alternative oxidase gene AOX1a and studied their viability under subzero temperature (-10 °С for 3, 6, 9 hours). Cell viability and reactive oxygen species (ROS) production were assessed using fluorescence microscopy with fluorescein diacetate (FDA) and propidium iodide (PI) for cell viability measurement and H2DCF-DA for ROS measurement. The proportion of living cells was calculated as the proportion of FDApositive and PI-negative cells. Differences between the studied lines were determined in the content of mitochondrial proteins of the respiratory chain (AOX, COXII, NDB) and uncoupling protein (UCP), as well as in the intensity of formation of ROS and frost resistance. The obtained results confirmed the higher content of the AOX protein and its high contribution to mitochondrial respiration in line XX-2. Suspension culture cells of the AS-12 line showed a decrease in the AOX protein content and its contribution to mitochondrial respiration, compared to the wild type (Col-0) and line XX-2. Simultaneously with a decrease in the AOX protein content in the AS-12 cell culture, an increase in the content of the uncoupling protein UCP and subunit II of cytochrome oxidase (COXII) was observed. ROS generation was reduced in cell cultures of both XX-2 and AS-12. The obtained results indicate that the cells of the wildtype (Col-0) suspension culture were subjected to the most significant effect of subzero temperature. Long-term exposure (for 9 h) under -10 °С revealed significant differences in the viability of wild-type culture cells and lines with altered AOX1a gene expression. Cells of line XX-2 with an increased content of AOX turned out to be more resistant to subzero temperature compared to wild-type and AS-12 cells. However, while the proportion of living cells in the culture of the AS-12 line 48 h after exposure remained at the same level as immediately after it, in the suspension culture of the wild type cell death developed over time. The obtained results indicate the importance of alternative oxidase in the development of frost resistance in plant cell.

Gene expression profiles during heat acclimation in Arabidopsis thaliana suspension-culture cells

2006 ◽  
Vol 119 (4) ◽  
pp. 373-383 ◽  
Author(s):  
Chan Ju Lim ◽  
Kyung Ae Yang ◽  
Joon Ki Hong ◽  
Jin Soo Choi ◽  
Dea-Jin Yun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Arabidopsis Thaliana ◽  
Suspension Culture ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Heat Acclimation ◽  
Culture Cells

scholarly journals Inhibition of cell expansion enhances cortical microtubule stability in the root apex of Arabidopsis thaliana

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Veronica Giourieva ◽  
Emmanuel Panteris
Keyword(s):  
Arabidopsis Thaliana ◽  
Cell Wall ◽  
Cell Expansion ◽  
Cortical Microtubule ◽  
Root Apex ◽  
Cellulose Synthase ◽  
Cellulose Biosynthesis ◽  
Cortical Microtubules ◽  
Wild Type ◽  
Microtubule Stability

Abstract Background Cortical microtubules regulate cell expansion by determining cellulose microfibril orientation in the root apex of Arabidopsis thaliana. While the regulation of cell wall properties by cortical microtubules is well studied, the data on the influence of cell wall to cortical microtubule organization and stability remain scarce. Studies on cellulose biosynthesis mutants revealed that cortical microtubules depend on Cellulose Synthase A (CESA) function and/or cell expansion. Furthermore, it has been reported that cortical microtubules in cellulose-deficient mutants are hypersensitive to oryzalin. In this work, the persistence of cortical microtubules against anti-microtubule treatment was thoroughly studied in the roots of several cesa mutants, namely thanatos, mre1, any1, prc1-1 and rsw1, and the Cellulose Synthase Interacting 1 protein (csi1) mutant pom2-4. In addition, various treatments with drugs affecting cell expansion were performed on wild-type roots. Whole mount tubulin immunolabeling was applied in the above roots and observations were performed by confocal microscopy. Results Cortical microtubules in all mutants showed statistically significant increased persistence against anti-microtubule drugs, compared to those of the wild-type. Furthermore, to examine if the enhanced stability of cortical microtubules was due to reduced cellulose biosynthesis or to suppression of cell expansion, treatments of wild-type roots with 2,6-dichlorobenzonitrile (DCB) and Congo red were performed. After these treatments, cortical microtubules appeared more resistant to oryzalin, than in the control. Conclusions According to these findings, it may be concluded that inhibition of cell expansion, irrespective of the cause, results in increased microtubule stability in A. thaliana root. In addition, cell expansion does not only rely on cortical microtubule orientation but also plays a regulatory role in microtubule dynamics, as well. Various hypotheses may explain the increased cortical microtubule stability under decreased cell expansion such as the role of cell wall sensors and the presence of less dynamic cortical microtubules.

scholarly journals Eicosapentaenoic Acid Increases Browning Markers in Subcutaneous Adipose Tissue and Primary Adipocytes from Wild Type and UCP-1 Deficient Mice (P15-007-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Shasika Jayarathne ◽  
Mandana Pahlavani ◽  
Latha Ramalingam ◽  
Shane Scoggin ◽  
Naima Moustaid-Moussa
Keyword(s):  
Adipose Tissue ◽  
Eicosapentaenoic Acid ◽  
Subcutaneous Adipose Tissue ◽  
Uncoupling Protein ◽  
Fibroblast Growth Factor 21 ◽  
Chow Diet ◽  
Mrna Levels ◽  
Wild Type ◽  
Brown Adipose ◽  
Subcutaneous Adipose

Abstract Objectives Brown adipose tissue (BAT) regulates energy balance through thermogenesis, in part via uncoupling protein -1 (UCP-1). White adipose tissue (WAT), namely subcutaneous adipose tissue (SAT) can convert to a beige/brite adipose tissue phenotype (browning) under thermogenic conditions such as cold. We previously reported that eicosapentaenoic acid (EPA) reduced obesity and glucose intolerance, and increased UCP-1 in BAT of B6 mice at ambient temperature (22°C); and these effects were attenuated at thermoneutral environment (28–30°C). We hypothesized that EPA exerts anti-obesity effects on SAT, including increased browning, adipocyte hypotrophy; and these effects require UCP-1. Methods Six-week-old B6 wild type (WT) and UCP-1 knock-out (KO) male mice were maintained at thermoneutral environment and fed high fat diet (HF) with or without 36 g/kg of AlaskOmega EPA-enriched fish oil (800 mg/g) for 14 weeks; and SAT was collected for histological, gene and protein analyses. SAT was also prepared from chow diet-fed WT and KO mice at ambient environment to prepare stroma vascular cells, which were differentiated into adipocytes, treated with 100uM EPA for 48 hours then harvested for mRNA and protein analyses. Results KO mice fed HF diets had the highest body weight (P < 0.05) among all groups. EPA reduced fat cell size in both WT and KO mice fed the EPA diet. mRNA levels of fibroblast growth factor-21 (FGF-21) were higher in SAT of WT mice fed EPA compared to WT mice fed HF (P < 0.05), with no differences between the KO genotype. KO mice fed HF diets had lower levels of UCP-3 in SAT compared to WT mice fed HF (P < 0.05), which was rescued only in the KO mice fed EPA (P < 0.05). UCP-1 protein levels were very low in SAT tissues, and UCP-2 mRNA levels were similar across all groups in SAT. Interestingly, EPA significantly (P < 0.05) increased mRNA expression of UCP-2, UCP-3 and FGF21 in differentiated SAT adipocytes from both WT and KO compared to control. Furthermore, UCP-1 mRNA levels were significantly higher in WT adipocytes treated with EPA, compared to non-treated cells (P < 0.05). Additional mechanistic studies are currently underway to further dissect adipose depot differences in EPA effects in WT vs. KO mice. Conclusions Our data suggest that EPA increases SAT browning, independently of UCP-1. Funding Sources NIH/NCCIH.

scholarly journals Mitochondrial Respiration in KRAS and BRAF Mutated Colorectal Tumors and Polyps

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 815 ◽  
Author(s):  
Egle Rebane-Klemm ◽  
Laura Truu ◽  
Leenu Reinsalu ◽  
Marju Puurand ◽  
Igor Shevchuk ◽  
...  
Keyword(s):  
Mitochondrial Respiration ◽  
Atp Synthesis ◽  
Colorectal Polyps ◽  
Metabolic Phenotype ◽  
Mitochondrial Outer Membrane ◽  
Control Group ◽  
Colon Polyps ◽  
Wild Type ◽  
Tissue Samples ◽  
Potential Component

This study aimed to characterize the ATP-synthesis by oxidative phosphorylation in colorectal cancer (CRC) and premalignant colon polyps in relation to molecular biomarkers KRAS and BRAF. This prospective study included 48 patients. Resected colorectal polyps and postoperative CRC tissue with adjacent normal tissue (control) were collected. Patients with polyps and CRC were divided into three molecular groups: KRAS mutated, BRAF mutated and KRAS/BRAF wild-type. Mitochondrial respiration in permeabilized tissue samples was observed using high resolution respirometry. ADP-activated respiration rate (Vmax) and an apparent affinity of mitochondria to ADP, which is related to mitochondrial outer membrane (MOM) permeability, were determined. Clear differences were present between molecular groups. KRAS mutated CRC group had lower Vmax values compared to wild-type; however, the Vmax value was higher than in the control group, while MOM permeability did not change. This suggests that KRAS mutation status might be involved in acquiring oxidative phenotype. KRAS mutated polyps had higher Vmax values and elevated MOM permeability as compared to the control. BRAF mutated CRC and polyps had reduced respiration and altered MOM permeability, indicating a glycolytic phenotype. To conclude, prognostic biomarkers KRAS and BRAF are likely related to the metabolic phenotype in CRC and polyps. Assessment of the tumor mitochondrial ATP synthesis could be a potential component of patient risk stratification.

scholarly journals Quintuple Deglycosylation Mutant of Simian Immunodeficiency Virus SIVmac239 in Rhesus Macaques: Robust Primary Replication, Tightly Contained Chronic Infection, and Elicitation of Potent Immunity against the Parental Wild-Type Strain

2001 ◽  
Vol 75 (9) ◽  
pp. 4023-4028 ◽  
Author(s):  
Kazuyasu Mori ◽  
Yasuhiro Yasutomi ◽  
Shinji Ohgimoto ◽  
Tadashi Nakasone ◽  
Shiki Takamura ◽  
...  
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Chronic Infection ◽  
Neutralizing Antibodies ◽  
Rhesus Macaques ◽  
Chronic Phase ◽  
Wild Type ◽  
Live Attenuated Vaccine ◽  
Challenge Virus ◽  
Culture Cells ◽  
Immunodeficiency Virus

ABSTRACT We previously generated a mutant of simian immunodeficiency virus (SIV) lacking 5 of a total of 22 N-glycans in its external envelope protein gp120 with no impairment in viral replication capability and infectivity in tissue culture cells. Here, we infected rhesus macaques with this mutant and found that it also replicated robustly in the acute phase but was tightly, though not completely, contained in the chronic phase. Thus, a critical requirement for the N-glycans for the full extent of chronic infection was demonstrated. No evidence indicating reversion to a wild type was obtained during the observation period of more than 40 weeks. Monkeys infected with the mutant were found to tolerate a challenge infection with wild-type SIV very well. Analyses of host responses following challenge revealed no neutralizing antibodies against the challenge virus but strong secondary responses of cytotoxic T lymphocytes against multiple antigens, including Gag-Pol, Nef, and Env. Thus, the quintuple deglycosylation mutant appeared to represent a novel class of SIV live attenuated vaccine.

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