Journal: Microorganisms

Article Title: Preclinical Testing of Boron-Doped Diamond Electrodes for Root Canal Disinfection—A Series of Preliminary Studies

doi: 10.3390/microorganisms10040782

Figure Lengend Snippet: Microscopic imaging of biofilm formation. The extracellular polysaccharide matrix of biofilm-forming microorganisms was stained using EbbaBiolight680 after 5 ( a , b ) and 7 ( c , d ) days of incubation. Biofilm was either removed from the root canals before staining using toothpicks ( a , c ) or split teeth were analyzed ( b , d ). Panel b shows biofilm in a side channel and panel d shows biofilm attached to the root canal wall located at the bottom of the picture. ( a – c ) are overlays of brightfield and fluorescence, ( d ) show fluorescence in the red channel of the microscope only.

Article Snippet: After thirty minutes of incubation, the samples were gently washed with water, dried and analyzed with a confocal laser scanning microscope (CLSM, Axio Vert.A1, Carl Zeiss Microscopy Deutschland GmbH, Oberkochen, Germany) using the following equipment: Objectives: A-Plan 10×/0.25 Ph 1; LD A-Plan 40×/0.55 Ph 1; EC Plan-Neofluar 63×/1.25 Oil M27; Channel: mRFP, bright; Camera: AxioCamICm1; Software: Zen 2.3 (blue edition, Version 2.3.69.1018).

Techniques: Imaging, Staining, Incubation, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Functional Characterization of fer-ts , a Temperature-Sensitive FERONIA Mutant Allele That Alters Root Hair Growth

doi: 10.1101/2020.05.27.119891

Figure Lengend Snippet: (a) Seven day-old seedling plants were grown vertically in 1/4 MS media under 20°C and transferred to 30°C for 6 h, followed by 24 h recovery at 20°C. Bright field images were collected with a Nikon Eclipse E600 wide-field microscope with a 10x Plan Apo DIC (0.75 NA) lens. Dashed lines indicate root tip positions when seedling plants were transferred to 30°C condition for 6 hours, and again when they were transferred back to 20°C. Scale bars = 200 μm. (b) Localization of EYFP-RabA4b protein in growing roothair cells of fer-ts mutants at 20°C and 30°C. Medial root hair sections were collected using spinning-disk confocal microscopy from growing root-hair cells of seven-day-old seedlings stably expressing EYFP-RabA4b in the fer-ts mutant in 20°C (left) or 30°C (right) using a Zeiss 40x Plan-Apochromat (1.3 NA) lens and appropriate EYFP fluorescence filter sets. Scale bars = 10 μm. Insets, magnified images to show details of EYFP-RabA4b subcellular localization in root-hair tips. Scale bars = 2 μm. (c) Quantification of root hair length in WT and fer-ts mutants under 20°C (wild-type (n=392), fer-ts (n=454)) and 30°C (wild-type (n=185), fer-ts (n=23)) conditions. (d) Calculation of root hair densities in WT and fer-ts mutants at 20°C (wild-type (n=392), fer-ts (n=454)) and 30°C (wild-type (n=185), fer-ts (n=23)) in fully expanded primary roots of seven-day old plants. In each case, root hair lengths and densities were measured from n=20 individual seedlings. Error bars represent SD. **p<0.001 by Student’s t -test.

Article Snippet: Confocal images were generated using a laser confocal microscope (Zeiss Observer.A1) connected to a CSU10 confocal scanner unit (Yokogawa, Japan) and a 10x Plan-Neofluar (0.3 NA lens), 40x Plan-Apochromat (1.3 NA lens) or 100x Plan-Apochromat (1.46 NA lens) oil objective with 491 nm laser excitation and a 535 nm emission filter for EGFP and EYFP fluorescence.

Techniques: Microscopy, Confocal Microscopy, Stable Transfection, Expressing, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: Functional Characterization of fer-ts , a Temperature-Sensitive FERONIA Mutant Allele That Alters Root Hair Growth

doi: 10.1101/2020.05.27.119891

Figure Lengend Snippet: Wild-type (WT), fer-ts , or fer-5 plants were germinated and grown for 3 days in ½ MS liquid media at 20°C, and then transferred to ½ MS liquid media containing 1uM RALF1 peptide (RALF+) or a mock buffer control (RALF-) and grown an additional 3 days at 20°C (a) or 30°C (b). Images of representative seedlings were collected using an Olympus SZX12 stereoscopic microscope. Quantification of primary root lengths (n = 10 seedlings) in the presence or absence of RALF1 peptide treatment in normal, 20°C (c) and elevated, 30°C (d) conditions. Primary root lengths were determined using Image J. Error bars represent SD. *p<0.05, **p<0.01 by Student’s t -test.

Article Snippet: Confocal images were generated using a laser confocal microscope (Zeiss Observer.A1) connected to a CSU10 confocal scanner unit (Yokogawa, Japan) and a 10x Plan-Neofluar (0.3 NA lens), 40x Plan-Apochromat (1.3 NA lens) or 100x Plan-Apochromat (1.46 NA lens) oil objective with 491 nm laser excitation and a 535 nm emission filter for EGFP and EYFP fluorescence.

Techniques: Control, Microscopy

Journal: bioRxiv

Article Title: Functional Characterization of fer-ts , a Temperature-Sensitive FERONIA Mutant Allele That Alters Root Hair Growth

doi: 10.1101/2020.05.27.119891

Figure Lengend Snippet: (a) Wild-type (WT), fer-4, fer-5 , and F1 progeny from crosses (paternal = fer-ts , maternal = fer-4 or fer-5 ) of fer-ts/fer-4 and fer-ts/fer-5 were grown vertically for seven days at 20°C, transferred to 30°C for 6 h, and then grown for an additional 24 h at 20°C. Both fer-ts/fer-4 and fer-ts/fer-5 progeny clearly demonstrated a ts-dependent root hair phenotype. Scale bars = 200 μm. (b) Schematic diagram of the FERONIA gene structure. Open and filled boxes indicate untranslated regions (UTRs) and exon regions, respectively. The locations of T-DNA insertion mutants ( fer-4 and fer-5 ) and fer-ts are indicated by triangles and arrows, respectively. (c) Genotyping of crossed F1 plants. Genomic DNA was extracted from F1 generation plants and subjected PCR to confirm presence of the fer-4 and fer-5 genotypes (d) Both fer-4 and fer-5 display temperature-dependent root hair phenotypes when transformed with a fluorescently-tagged FER construct containing the fer-ts mutation (pFER-FER(G41S)-EYFP). Seven-day old seedlings stably transformed and homozygous for (pFER-EYFP(G41S)-EYFP) were grown vertically at 20°C, transferred to 30°C for 6 h, and then grown for an additional 24 h at 20°C. Presence of the transgenic pFER-FER(G41S)-EYFP construct clearly demonstrated a ts-dependent root hair phenotype. Scale bars = 200 μm. (e) Temperature sensitive root hair growth defects of fer-ts are fully rescued when transformed with a fluorescently-tagged FER construct (pFER-FER(WT)-EYFP). Seven-day old seedlings stably transformed and homozygous for (pFER-EYFP(WT)-EYFP)were grown vertically at 20°C, transferred to 30°C for 6 h, and then grown for an additional 24 h at 20°C. Bright field images were collected with a Nikon Eclipse E600 wide-field microscope with a 10x Plan Apo DIC (0.75 NA) lens. (f) Subcellular localization of FER(WT)-EYFP protein in roots and root hairs in a rescued fer-ts mutant plant. Fluorescent confocal images displaying the subcellular distribution of FER(WT)-EYFP protein was detected from growing root, and mature root hair cells of seven-day-old fer-ts seedlings stably transformed and homozygous for pFER-FER(WT)-EYFP. Cells were counter-stained by incubating for 5 min in a FM4-64 to visualize cell membranes. Images were collected by spinning-disk fluorescence confocal microscopy using a Zeiss 40x Plan-Apochromat (1.3 NA) lens with appropriate EYFP and FM4-64 fluorescence filter sets. Scale bars = 20 μm.

Article Snippet: Confocal images were generated using a laser confocal microscope (Zeiss Observer.A1) connected to a CSU10 confocal scanner unit (Yokogawa, Japan) and a 10x Plan-Neofluar (0.3 NA lens), 40x Plan-Apochromat (1.3 NA lens) or 100x Plan-Apochromat (1.46 NA lens) oil objective with 491 nm laser excitation and a 535 nm emission filter for EGFP and EYFP fluorescence.

Techniques: Transformation Assay, Construct, Mutagenesis, Stable Transfection, Transgenic Assay, Microscopy, Staining, Fluorescence, Confocal Microscopy

Journal: bioRxiv

Article Title: Functional Characterization of fer-ts , a Temperature-Sensitive FERONIA Mutant Allele That Alters Root Hair Growth

doi: 10.1101/2020.05.27.119891

Figure Lengend Snippet: (a) Root hair tip-growth in stably transformed lines expressing FER(WT)-EYFP and FER(G41S)-EYFP fluorescent fusion proteins under (20°C) and (30°C) temperature by time-lapse microscopy. Confocal images of were acquired using a Leica confocal laser-scanning microscope SP8 with a 63x oil lens (Numerical Aperture = 1.4) with 5s intervals. Representative images are presented at 50s intervals (10 frames). Scale bar: 10 μm. (b) Schematic diagram shown areas and lines used for fluorescence intensity quantification. Apical (red line) and peripheral (blue line) plasma membrane domains were measured, and the area between the apical plasma membrane and green line represents the vesicle-rich zone. (c) Quantitative analysis of root hair growth in stably transformed lines expressing FER(WT)-EYFP (n=3) and FER(G41S)-EYFP (n=3). Root hair elongation was measured every 50s using the measurement function in ImageJ. (d) Quantitative analysis of YFP intensity of vesicle-rich zone. (e) Quantitative analysis of YFP intensity of apical/ peripheral ratio. Values are normalized using 0s as 100%. The dashed line indicates time point of transition from permissive (20°C) to (30°C). Error bars in (c, d, e) represent SD. (f) Protein turnover rates of FER(WT)-EYFP and FER(G41S)-EYFP at elevated temperature (30°C). Five-day old seedling were grown at 20°C and then treated with 200 μM cycloheximide and transferred to 30°C. Total proteins were extracted at each time point and the relative levels were determined using immunoblotting with anti-GFP and anti-actin antibodies. FER(G41S)-EYFP levels rapidly decreased during the time course, while levels of FER(WT)-EYFP were not significantly reduced. Actin was used as a loading control.

Article Snippet: Confocal images were generated using a laser confocal microscope (Zeiss Observer.A1) connected to a CSU10 confocal scanner unit (Yokogawa, Japan) and a 10x Plan-Neofluar (0.3 NA lens), 40x Plan-Apochromat (1.3 NA lens) or 100x Plan-Apochromat (1.46 NA lens) oil objective with 491 nm laser excitation and a 535 nm emission filter for EGFP and EYFP fluorescence.

Techniques: Stable Transfection, Transformation Assay, Expressing, Time-lapse Microscopy, Laser-Scanning Microscopy, Fluorescence, Clinical Proteomics, Membrane, Western Blot, Control

Journal: bioRxiv

Article Title: Functional Characterization of fer-ts , a Temperature-Sensitive FERONIA Mutant Allele That Alters Root Hair Growth

doi: 10.1101/2020.05.27.119891

Figure Lengend Snippet: (a) Root hair tip-growth in stably transformed lines expressing FER(WT)-EYFP fluorescent fusion protein under permissive (20°C) temperature by time-lapse microscopy. Bright field images of growing root hair cells (n=3) were collected using a Nikon Eclipse E600 wide-field microscope with a x 20 Plan Apo (0.45 NA) lens with 5s intervals. The dashed line represents when media containing RALF1 (1 μM) was perfused into the growth chamber. Representative images are presented at 200s intervals. Scale bar: 50 μm. (b) Quantification of root hair growth. Root hair elongation was measured every 50s using the measurement function in ImageJ. Error bars represent SD.

Article Snippet: Confocal images were generated using a laser confocal microscope (Zeiss Observer.A1) connected to a CSU10 confocal scanner unit (Yokogawa, Japan) and a 10x Plan-Neofluar (0.3 NA lens), 40x Plan-Apochromat (1.3 NA lens) or 100x Plan-Apochromat (1.46 NA lens) oil objective with 491 nm laser excitation and a 535 nm emission filter for EGFP and EYFP fluorescence.

Techniques: Stable Transfection, Transformation Assay, Expressing, Time-lapse Microscopy, Microscopy

Journal: bioRxiv

Article Title: Functional Characterization of fer-ts , a Temperature-Sensitive FERONIA Mutant Allele That Alters Root Hair Growth

doi: 10.1101/2020.05.27.119891

Figure Lengend Snippet: (a) ROS accumulation in normal and elevated temperature conditions with or without auxin treatments. Wild-type (WT), fer-ts , fer-4 , or fer-5 seedlings were grown vertically on ¼ MS media plates for seven days at normal (20°C) or elevated (30°C) temperatures in the presence or absence of (10 nM NAA). Plates were bathed with five ml of 50 uM in H 2 DCF-DA suspended in ¼ MS liquid media for 5 min, followed by two gentle washes with 10 ml of ¼ MS. Fluorescence images were collected with a Zeiss Axio Imager Z1 fluorescence microscope with 2.5x objective and green (GFP) filter set. The WT ROS image was acquired by auto-exposure, all other images were acquired using the WT exposure conditions. Scale bars = 500 μm. (b) The rectangle in (a) indicates a representative region of interest (ROI) where average ROS intensity was quantified for the samples. Intensities of ROS were quantified by image J program. Error bars represent SD. **p<0.01 by Student’s t -test.

Article Snippet: Confocal images were generated using a laser confocal microscope (Zeiss Observer.A1) connected to a CSU10 confocal scanner unit (Yokogawa, Japan) and a 10x Plan-Neofluar (0.3 NA lens), 40x Plan-Apochromat (1.3 NA lens) or 100x Plan-Apochromat (1.46 NA lens) oil objective with 491 nm laser excitation and a 535 nm emission filter for EGFP and EYFP fluorescence.

Techniques: Gentle, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Functional Characterization of fer-ts , a Temperature-Sensitive FERONIA Mutant Allele That Alters Root Hair Growth

doi: 10.1101/2020.05.27.119891

Figure Lengend Snippet: (a and b) Quantification of root hair length and density in normal (upper panels) and elevated temperature (lower panels) in 10, 100, and 1000 uM concentrations for each of the various hormone treatments (NAA; Auxin, ACC; ethylene, Kinetin; Cytokinin, ABA; Abscisic acid, eBL; epi-brassinosteroid, MeJA; methyl jasmonic acid, SA; Salicylic acid). Three-day-old seedlings were transferred into ½ MS liquid media containing three different concentrations of the various hormones. Then, transferred plants were incubated at 20°C and 30°C for seven days before quantification. The root hairs were photographed by Olympus AX-70 microscope and root hair length and densities were determined by image J. Error bars represent SD.

Article Snippet: Confocal images were generated using a laser confocal microscope (Zeiss Observer.A1) connected to a CSU10 confocal scanner unit (Yokogawa, Japan) and a 10x Plan-Neofluar (0.3 NA lens), 40x Plan-Apochromat (1.3 NA lens) or 100x Plan-Apochromat (1.46 NA lens) oil objective with 491 nm laser excitation and a 535 nm emission filter for EGFP and EYFP fluorescence.

Techniques: Incubation, Microscopy