Journal: bioRxiv

Article Title: Mitochondria affect photosynthesis through altered tissue levels of oxygen

doi: 10.1101/2024.07.19.604342

Figure Lengend Snippet: Representative anti-AOX1 immunoblot and corresponding total protein abundance (A). Inhibition of O 2 consumption after addition of inhibitors KCN and SHAM (B), shown values averages +/-standard deviations.

Article Snippet: An O 2 microsensor (OX-10, Unisense, Denmark) was inserted 80-100 µm into the main vein of the leaf lamina, with 0 indicating the tissue surface.

Techniques: Western Blot, Inhibition

Journal: bioRxiv

Article Title: Mitochondria affect photosynthesis through altered tissue levels of oxygen

doi: 10.1101/2024.07.19.604342

Figure Lengend Snippet: AOX abundance was measured by immunoblotting total protein extracts from seedlings grown on plates (A), and AOX capacity was determined as the fraction of O 2 consumption that was not inhibited by KCN (B). The genotypes are arranged in order of increasing mean value. Quantification in (A) was done using 4 independent immunoblots, and in (B) with 6 independent biological replicates. Statistically significant groups are indicated with letters, which are bolded for lines that have statistically significant difference from wild type. There was a strong correlation between AOX abundance and capacity (C), with Pearson correlation value of 0.8704 and p-value < 0.0001. The plant lines selected for in-depth analyses (see below) are marked with separately colored dots.

Article Snippet: An O 2 microsensor (OX-10, Unisense, Denmark) was inserted 80-100 µm into the main vein of the leaf lamina, with 0 indicating the tissue surface.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Mitochondria affect photosynthesis through altered tissue levels of oxygen

doi: 10.1101/2024.07.19.604342

Figure Lengend Snippet: O 2 dynamics in selected plant lines were examined by measuring O 2 fluctuations in cuvettes where aerial parts of 3-week-old plate-grown plants were immersed in analysis buffer. An example of this assay performed with Col-0 and rcd1 is shown in (A), with relative O 2 consumption rate first recorded in darkness. After O 2 concentrations approached zero, the light was turned on (yellow vertical line) and O 2 re-accumulation due to photosynthetic O 2 evolution was recorded. Consumption (B) and re-accumulation (C) rates of O 2 were obtained from the kinetics as shown in (A). The genotypes are arranged in order of increasing mean value. Statistically significant groups are indicated with letters, which are bolded for lines that have higher O 2 consumption or lower O 2 re-accumulation rates than wild type.

Article Snippet: An O 2 microsensor (OX-10, Unisense, Denmark) was inserted 80-100 µm into the main vein of the leaf lamina, with 0 indicating the tissue surface.

Techniques:

Journal: bioRxiv

Article Title: Mitochondria affect photosynthesis through altered tissue levels of oxygen

doi: 10.1101/2024.07.19.604342

Figure Lengend Snippet: Leaves of Col-0 and rcd1 plants were inserted into deoxygenated medium and probed with microsensors. A microscope image of the microsensor being inserted into the leaf is shown in (A). Rates for O 2 consumption (in darkness) and recovery (in light) were measured from inside the central vein of the leaf (B). Statistically significant groups are indicated with letters.

Article Snippet: An O 2 microsensor (OX-10, Unisense, Denmark) was inserted 80-100 µm into the main vein of the leaf lamina, with 0 indicating the tissue surface.

Techniques: Microscopy

Journal: bioRxiv

Article Title: Mitochondria affect photosynthesis through altered tissue levels of oxygen

doi: 10.1101/2024.07.19.604342

Figure Lengend Snippet: Parameters that were calculated in this study were analyzed using Pearson correlation. Correlations were calculated in tests where all available plant lines were used (A), or with atypically behaving AOX1a- OE line excluded (B). A separate matrix was calculated for the selected lines tested in the O 2 consumption / re-accumulation assay (C). Plant lines included in (A), (B) and (C) are shown in (D). Size and color of the circles indicate strength of correlation, the color scale is shown to the right of each panel. Statistically nonsignificant correlations (p > 0.05) are marked with crosses. For more detail see Supplementary Dataset 1. Raw data and statistics.

Article Snippet: An O 2 microsensor (OX-10, Unisense, Denmark) was inserted 80-100 µm into the main vein of the leaf lamina, with 0 indicating the tissue surface.

Techniques:

Journal: bioRxiv

Article Title: Mycobacterium tuberculosis H 2 S functions as a sink to modulate central metabolism, bioenergetics, and drug susceptibility

doi: 10.1101/2021.06.02.446746

Figure Lengend Snippet: (a) Detection of H 2 S in the headspace of cultures of mycobacterial species using lead acetate strips. Msm ; M. smegmatis , BCG; M. bovis BCG, Mbo ; M. bovis , Mtb ; Mtb H37Rv, CDC; Mtb CDC1551, MDR; multi-drug resistant and DS; drug-sensitive clinical strains of Mtb . Note: Strips shown, ( top insert) were scanned after 48 h of incubation. The inkwell bottles shown are representative of an independent experiment after 72 h of incubation. (b) Estimation of H 2 S production by quantifying lead sulfide staining ( top insert) using densitometric analysis. Data normalized to the optical density (OD 600 ) of each culture; n = 3. (c) Microplate-based BC assay showing H 2 S production by live and heat-killed Mtb H37Rv. Time course measurement of H 2 S production using BC assay for (d) intact Mtb H37Rv cells, ( n = 5 – 8) and (e) lysates with different concentrations of Cys, ( n = 3 – 4). BC assay of (f) intact Mtb H37Rv cells, ( n = 3 – 5) and (g) Mtb H37Rv lysates in the presence of AOAA showing reduced H 2 S production, ( n = 4). No significant inhibition of H 2 S production for (h) intact Mtb H37Rv cells, ( n = 3 – 4) and (i) lysates was demonstrated in the presence of PAG, ( n = 4). A Unisense A/S H 2 S microsensor was used to measure the H 2 S concentration in (j) Mtb H37Rv cultures and bacteria-free supernatants, ( n = 4). Microsensor measurements of real-time H 2 S production in Mtb H37Rv lysates in assay buffer (0.4 M triethanolamine-HCl, pH 8.0; 20 µM PLP, 10 mM EDTA) with (k) 20 mM Cys, (l) stepwise addition of Cys (arrows indicate the amount of Cys (mM) added at the time point) and (m) 20 mM Cys followed by the addition of Mtb lysate preincubated with AOAA (4 mM). Representative experiments are shown in each panel, each experiment was repeated at least twice. Data shown represent the mean ± SD from the indicated n (number of replicates per data set) except for ( K , L and M ), which show representative real-time measurement for one sample with 2-3 independent repeats. All P values are relative to untreated controls or as indicated. Statistical analysis was performed using GraphPad Prism 7.02. One-way ANOVA with Dunnett’s multiple comparisons test was used to determine statistical significance. * P < 0.05, ** P < 0.01, *** P < 0.001, # P < 0.0001.

Article Snippet: To further support these findings, we used a highly sensitive amperometric H 2 S microsensor (Unisense, A/S, Denmark) to directly detect H 2 S. Using this method, we observed that, compared to untreated controls, the addition of 1 mM Cys resulted in a 3-fold increase in H 2 S levels in the media of dispersed Mtb cultures, or in the corresponding cleared media containing pelleted cells after 72 hours of incubation ( ).

Techniques: Incubation, Staining, Inhibition, Concentration Assay

Journal: bioRxiv

Article Title: Mycobacterium tuberculosis H 2 S functions as a sink to modulate central metabolism, bioenergetics, and drug susceptibility

doi: 10.1101/2021.06.02.446746

Figure Lengend Snippet: Mtb respiration was measured using an Agilent Seahorse XF96 Analyzer. (a) An OCR profile showing the basal respiration of Mtb strains, ( n = 5 - 7) (b) %OCR of Mtb upon addition of Cys, ( n = 3 - 5) (c) Fold OCR of Mtb strains relative to media control in the presence of 4 mM Cys, ( n = 3 - 7) (d) %OCR of Mtb with sequential injection of Cys and AOAA (1mM), or media (Med) as a control, ( n = 3) and (e) Measurement of H 2 S concentration in an OASS enzymatic activity assay using the Unisense H 2 S microsensor. H 2 S levels rapidly diminished after addition of OASS and substrate O -acetyl-L-serine (OAS) (f) %OCR of Mtb after injecting either Cys or OAS or Cys, OAS and OASS, ( n = 3 - 4). (g) %OCR of Mtb after injecting either Cys or Cys and OAS, ( n = 3 - 4). (h) Model showing how H 2 S stimulates respiration via both oxidases (dotted line arrows) and how respiration was reduced by exogenous addition of the H 2 S consuming enzyme OASS, and endogenous CysK1. Red “X”; inhibition, blue arrow; increased OCR. (i) Model showing electron flow through Complex I to the menaquinone pool (MK) and then through Complex III/IV (cytochrome bc 1 /aa 3 ), or re-routing of electrons through cytochrome bd if Complex III/IV is inhibited by Q203. This contributes to the proton-motive force, which powers ATP synthesis by Complex V (ATP synthase). OCR profiles of WT Mtb ( j, l ) or Δ cydAB cells ( k, m ) exposed to Cys, or Cys and Q203 (300x MIC 50 ), ( n = 3). Representative experiments are shown in each panel, each experiment was repeated at least twice. Data shown represent the mean ± SEM or ± SD (panel a ) from the indicated n (number of replicates per data set). Statistical analysis was performed using GraphPad Prism 7.02. One-way ANOVA with Dunnett’s multiple comparisons test (unpaired t-test for panel d ) was used to determine statistical significance. * P < 0.05, ** P < 0.01, *** P < 0.001, # P < 0.0001.

Article Snippet: To further support these findings, we used a highly sensitive amperometric H 2 S microsensor (Unisense, A/S, Denmark) to directly detect H 2 S. Using this method, we observed that, compared to untreated controls, the addition of 1 mM Cys resulted in a 3-fold increase in H 2 S levels in the media of dispersed Mtb cultures, or in the corresponding cleared media containing pelleted cells after 72 hours of incubation ( ).

Techniques: Injection, Concentration Assay, Enzyme Activity Assay, Inhibition