Journal: Plant Physiology

Article Title: Transcription Factor OsTGA10 Is a Target of the MADS Protein OsMADS8 and Is Required for Tapetum Development ^{1 [OPEN]}

doi: 10.1104/pp.17.01419

Figure Lengend Snippet: Characterization of OsTGA10 as a transcription factor. A, Subcellular localization of OsTGA10 in rice protoplasts. Top, localization of OsTGA10 fused with GFP. Bottom, localization of signal from empty vector containing GFP alone. B, Transcriptional activity assay in yeast. Full-length OsTGA10 amino acid sequence was fused with the DNA binding domain in pGBKT7. Yeast cells coexpressing pGBKT7-OsTGA10 and pGADT7 grew normally on selective medium lacking Leu, Trp, His, and Ade. Yeast cotransformed with pGBKT7-53 and pGADT7 served as a positive control, and yeast cotransformed with pGBKT7-Lam and pGADT7 served as a negative control. C, Schematic diagram showing the constructs used in the transient expression assays in D. D, Transcriptional activity assay in rice protoplasts. Compared to the negative control (GAL4BD), OsTGA10-GAL4BD significantly up-regulated the LUC/REN ratio. Error bars indicate the sd of four biological replicates. Student’s paired t test: *P < 0.05.

Article Snippet: Plant Materials and Growth Conditions Seeds of the T-DNA insertion mutant ostga10 (2A-10206L) and its corresponding wild type (Dong Jing) were obtained from the Postech Rice mutant database ( Yi and An, 2013 ).

Techniques: Plasmid Preparation, Activity Assay, Sequencing, Binding Assay, Positive Control, Negative Control, Construct, Expressing

Journal: Plant Physiology

Article Title: Transcription Factor OsTGA10 Is a Target of the MADS Protein OsMADS8 and Is Required for Tapetum Development ^{1 [OPEN]}

doi: 10.1104/pp.17.01419

Figure Lengend Snippet: Expression pattern of OsTGA10. A to H, Detection of OsTGA10 via in situ hybridization in wild-type anthers before meiosis stage. The anther developmental process is described in detail in Supplemental Figure S6. The anther sections were hybridized with antisense probe (A–F) or sense probe (G and H). A, Anther at stage 1; B, anther at stage 2; C, anther at stage 3; D and G, anther at stage 4; E and H, anther at stage 5; F, anther at stage 6-1. Bar = 50 μm. I to L, Magnified view of boxes in C to F. M to R, Detection of OsMADS8 via in situ hybridization in wild-type anthers before meiosis stage. M, anther at stage 1; N anther at stage 2; O anther at stage 3; P, anther at stage 4; Q, anther at stage 5; R, anther at stage 6-1. Bar = 50 μm. MMC, Microspore mother cell; PPC, primary parietal cell; SPC, secondary parietal cell; T, tapetum.

Article Snippet: Plant Materials and Growth Conditions Seeds of the T-DNA insertion mutant ostga10 (2A-10206L) and its corresponding wild type (Dong Jing) were obtained from the Postech Rice mutant database ( Yi and An, 2013 ).

Techniques: Expressing, In Situ Hybridization

Journal: Plant Physiology

Article Title: Transcription Factor OsTGA10 Is a Target of the MADS Protein OsMADS8 and Is Required for Tapetum Development ^{1 [OPEN]}

doi: 10.1104/pp.17.01419

Figure Lengend Snippet: Comparison between wild-type and ostga10 phenotypes. A, Comparison of wild-type (WT) and ostga10 mutant plants after seed maturation. Bar = 10 cm. B, Panicles in wild-type and ostga10 mutant plants. Bar = 10 cm. C and D, Florets in wild-type (C) and ostga10 mutant (D) plants. Half of the lemma and palea were removed. Bar = 500 μm. E and F, Scanning electron microscopy observation of wild-type (E) and ostga10 (F) anthers. Bar = 200 μm. G and H, Wild-type (G) and ostga10 (H) pollen grains were stained by I2-KI solution. Bar = 100 μm. I to L, Statistical analysis of seed setting (I), pollen grains (J and K), and abnormal anthers (L) in 24 panicles from eight independent ostga10 mutant lines and seven panicles from four independent wild-type lines.

Article Snippet: Plant Materials and Growth Conditions Seeds of the T-DNA insertion mutant ostga10 (2A-10206L) and its corresponding wild type (Dong Jing) were obtained from the Postech Rice mutant database ( Yi and An, 2013 ).

Techniques: Comparison, Mutagenesis, Electron Microscopy, Staining

Journal: Plant Physiology

Article Title: Transcription Factor OsTGA10 Is a Target of the MADS Protein OsMADS8 and Is Required for Tapetum Development ^{1 [OPEN]}

doi: 10.1104/pp.17.01419

Figure Lengend Snippet: Observation of sections representing wild-type and ostga10 anther development. The anther developmental process was described in detail in Supplemental Figure S6. The transverse sections from the middle of anthers were observed. A to H, Paraffin sections of wild-type (A–D) and ostga10 (E–H) anthers before meiosis stage. A and E, Anthers at stage 3; B and F, anthers at stage 4; C and G, anthers at stage 5; D and H, anthers at stage 6-1. Bar = 50 μm. I to P, Semithin sections of wild-type (I–L) and ostga10 (M–P) anthers after meiosis stage. I and M, Anthers at stage 7; J and N, anthers at stage 8; K and O, anthers at stage 9; L and P, anthers at stage 11. Bar = 20 μm; E, Epidermis; En, endothecium; M, middle layer; MC, meiotic cell; MMC, microspore mother cell; MP, mature pollen; Msp, microspore; PPC, primary parietal cell; Sp, sporogenous cells; SPC, second parietal cells; T, tapetum; Td, tetrads.

Article Snippet: Plant Materials and Growth Conditions Seeds of the T-DNA insertion mutant ostga10 (2A-10206L) and its corresponding wild type (Dong Jing) were obtained from the Postech Rice mutant database ( Yi and An, 2013 ).

Techniques:

Journal: Plant Physiology

Article Title: Transcription Factor OsTGA10 Is a Target of the MADS Protein OsMADS8 and Is Required for Tapetum Development ^{1 [OPEN]}

doi: 10.1104/pp.17.01419

Figure Lengend Snippet: TEM analysis of wild-type and ostga10 anther development. A, C, and I, Cross sections of wild-type anthers at stage 7 (A), stage 8 (C), and stage 9 (I). Bar = 5 μm. B, D, and J, Magnifications of tapetal cells highlighted by boxes in A, C, and I. Bar = 1 μm. E, G, and K, Cross sections of ostga10 anthers at stage 7 (E), stage 8 (G), and stage 9 (K). Bar = 5 μm. F, H, and L, Magnifications of tapetal cells highlighted by boxes in E, G, and K. Bar = 1 μm. Dashed boxes in J and L indicate ubisch bodes in tapetal cells. E, Epidermis; En, endothecium; ER, endoplasmic reticulum; M, middle layer; Msp, microspore; Mt, mitochondria; Nu, nucleus; T, tapetum; Ub, ubisch bodies.

Article Snippet: Plant Materials and Growth Conditions Seeds of the T-DNA insertion mutant ostga10 (2A-10206L) and its corresponding wild type (Dong Jing) were obtained from the Postech Rice mutant database ( Yi and An, 2013 ).

Techniques:

Journal: Plant Physiology

Article Title: Transcription Factor OsTGA10 Is a Target of the MADS Protein OsMADS8 and Is Required for Tapetum Development ^{1 [OPEN]}

doi: 10.1104/pp.17.01419

Figure Lengend Snippet: Comparison of DNA fragments in the tapetum of wild-type and ostga10 anthers. A to D, DNA fragments in the tapetum of wild-type anthers. Bars = 50 μm. E to H, DNA fragments in the tapetum of ostga10 anthers. Bars = 50 μm. A and E, Anthers at stage 6-2. B and F, Anthers at stage 7. C and G, Anthers at stage 8. D and H, Anthers at stage 9. The yellow fluorescence indicates TUNEL-positive signal, which is merged with red fluorescence from background staining and green fluorescence from TUNEL positive nuclei staining. Arrowheads indicate tapetal cells.

Article Snippet: Plant Materials and Growth Conditions Seeds of the T-DNA insertion mutant ostga10 (2A-10206L) and its corresponding wild type (Dong Jing) were obtained from the Postech Rice mutant database ( Yi and An, 2013 ).

Techniques: Comparison, Fluorescence, TUNEL Assay, Staining

Journal: Plant Physiology

Article Title: Transcription Factor OsTGA10 Is a Target of the MADS Protein OsMADS8 and Is Required for Tapetum Development ^{1 [OPEN]}

doi: 10.1104/pp.17.01419

Figure Lengend Snippet: RT-qPCR analysis of expression levels of tapetum genes in ostga10. S5 to S10 represent stages 5 to 10 of anther development (see Supplemental Fig. S6). Error bars indicate the SD of three biological replicates. Student’s paired t test: *P < 0.05, **P < 0.01.

Article Snippet: Plant Materials and Growth Conditions Seeds of the T-DNA insertion mutant ostga10 (2A-10206L) and its corresponding wild type (Dong Jing) were obtained from the Postech Rice mutant database ( Yi and An, 2013 ).

Techniques: Quantitative RT-PCR, Expressing

Journal: Plant Physiology

Article Title: Transcription Factor OsTGA10 Is a Target of the MADS Protein OsMADS8 and Is Required for Tapetum Development ^{1 [OPEN]}

doi: 10.1104/pp.17.01419

Figure Lengend Snippet: Interaction between OsTGA10 and TIP2 and between OsTGA10 and TDR. A, Yeast two-hybrid assay test for interaction. Yeast cells cotransformed with different constructs were grown on selective medium lacking Leu, Trp, His, and Ade. 2.5 mm and 5 mm 3-amino-1,2,4-triazole were added to inhibit self-transcriptional activation of OsTGA10. B, In vitro pull-down assay test for interaction. OsTGA10 fused with Trigger Factor (TF), TIP2 fused with GST, and TDR fused with GST were expressed in E. coli. Interactions were determined by immnoblot analysis using anti-TF antibody. Lanes without interactions between OsTGA10 and other proteins were not included in this immunoblot image. C, Split-luciferase complementation assay test for interaction in N. benthamiana leaf tissue. Luciferase signals were detected in leaf cells 48 h following coinfiltration with OsTGA10-nLuc and TIP2-cLuc and with OsTGA10-nLuc and TDR-cLuc. nLuc represents the N-terminal fragment of luciferase, whereas cLuc represents the C-terminal fragment of luciferase.

Article Snippet: Plant Materials and Growth Conditions Seeds of the T-DNA insertion mutant ostga10 (2A-10206L) and its corresponding wild type (Dong Jing) were obtained from the Postech Rice mutant database ( Yi and An, 2013 ).

Techniques: Y2H Assay, Construct, Activation Assay, In Vitro, Pull Down Assay, Western Blot, Luciferase

Journal: Plant Physiology

Article Title: Transcription Factor OsTGA10 Is a Target of the MADS Protein OsMADS8 and Is Required for Tapetum Development ^{1 [OPEN]}

doi: 10.1104/pp.17.01419

Figure Lengend Snippet: Analysis of genes bound by OsTGA10. A, Distribution of E-box and TGACG motifs in AP25 and MTR1 promoter sequences. B and C, EMSA analysis showed interaction between recombinant OsTGA10 and biotin-labeled probes containing the TGACG motif in the promoter of AP25 (B) and MTR1 (C). Unlabeled probes were used for competition. Arrows indicate the shifted bands and free probes. D, Schematic diagram depicting the constructs used in transient expression assays in E and F. E, The transcriptional repression of the AP25 promoter by OsTGA10 in N. benthamiana leaves. F, OsTGA10 further enhanced the transcriptional repression of the MTR1 promoter by TIP2 in N. benthamiana leaves. Error bars in E and F indicate the SD of six biological replicates. Student’s paired t test: *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Plant Materials and Growth Conditions Seeds of the T-DNA insertion mutant ostga10 (2A-10206L) and its corresponding wild type (Dong Jing) were obtained from the Postech Rice mutant database ( Yi and An, 2013 ).

Techniques: Recombinant, Labeling, Construct, Expressing

Journal: Plant Physiology

Article Title: Transcription Factor OsTGA10 Is a Target of the MADS Protein OsMADS8 and Is Required for Tapetum Development ^{1 [OPEN]}

doi: 10.1104/pp.17.01419

Figure Lengend Snippet: Defective endothecium secondary cell wall thickening in ostga10 anthers. A, C, E, and G, Sections of wild-type (A and C) and ostga10 (E and G) anthers. A and E, Anthers at stage 9, and C and G, anthers at stage 11. Bar = 20 μm. B, D, F, and H, The magnified views of anthers in A, C, E, and G illuminated by UV light. Arrowheads in B and D indicate signals of secondary cell wall thickening in endothecium cells. I, RT-qPCR analysis of expression levels of genes involved in rice secondary cell wall thickening in ostga10. Error bars indicate SD of three biological repeats. Student’s paired t test: *P < 0.05, **P < 0.01.

Article Snippet: Plant Materials and Growth Conditions Seeds of the T-DNA insertion mutant ostga10 (2A-10206L) and its corresponding wild type (Dong Jing) were obtained from the Postech Rice mutant database ( Yi and An, 2013 ).

Techniques: Quantitative RT-PCR, Expressing

Journal: Aging Cell

Article Title: FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16 INK4a increase

doi: 10.1111/acel.13226

Figure Lengend Snippet: FOXO3 and p16 INK4a oppositely modulate the vulnerability of human HD NSCs. Significance was tested using two‐way ANOVA (panels a–c), paired t test (panels d) and Mann‐Whitney test (panel g). ns: not significant. (A) Human HD NSCs show reduced rates of cell growth. Data are mean ± SEM . (b) Reducing FOXO3 does not alter the growth of C116 NSCs (left panel) and strongly reduces the growth of HD NSCs (right panel), with no change detected in HTT mRNA levels (see Figure , left panel). Data are mean ± SEM . (c) Reducing p16 INK4a slightly increases the growth of C116 (left panel) and HD (right panel) NSCs. Reducing p16 INK4a does not alter HTT mRNA levels in HD NSCs (see Figure , right panel). Data are mean ± SEM . (d) Reducing FOXO3 increases the mortality of HD NSCs with no effect detected in C116 NSCs (left: * p < .05). Reducing p16 INK4a decreases the mortality of HD NSCs with no effect detected in C116 NSCs (right: * p < .05). (e) Lenti‐myc‐ p16 INK4a transduction promotes nuclear release of HMGB1 in cytoplasm of HD and corrected (C116) MSNs. HD and C116 MSNs transduced for 4 days with lenti‐myc‐ p16 INK4a (red) were immunostained with HMGB1 (green). NT: transduction without myc‐ p16 INK4a . HMGB1 co‐localizes with the nucleus (DAPI), with low level in cytoplasm. The transduction with lenti‐myc‐ p16 INK4a significantly relocates HMGB1 into cytoplasm of HD and C116 MSNs (arrowhead). Scale bars: 100 µm. (f) Upper panel: The quantification of cytoplasmic HMGB1 pixel intensity shows a significant increase of nuclear HMGB1 release in HD vs. C116 MSNs and in HD vs. C116 MSNs following p16 INK4a overexpression (Wilcoxon ranked‐sum test: C116‐ p16 INK4a vs. HD‐ p16 INK4a , p = 6.1e‐22; C116‐ p16 INK4a vs. HD‐NT, p = 3.4e‐06; C116‐ p16 INK4a vs. C116‐NT, p = 1.8e‐23; HD‐ p16 INK4a vs. HD‐NT, p = 2.3e‐7; HD‐NT vs. C116‐NT, p = 5.6e‐37). Lower panel: data normalized against Myc‐p16 INK4a levels using the ratio (sum of HMGB1 intensity in ‘cells’ MOI 1/number of cells detected in cells MOI 1)/(sum of myc‐tag intensity in ‘cells’ MOI 1/number of cells detected in ‘cells’ MOI 1). The ratios show that HMGB1 relocalization is CAG‐repeat‐length‐dependent. C116‐p16 INK4a : 1604 cells; C116‐NT: 1403 cells; HD‐p16 INK4a : 879 cells; HD‐NT: 1792 cells

Article Snippet: Lentivirus transduction was performed with myc‐p16 INK4a from Origene (RC220937L1V) using a multiplicity of infection (MOI) of 1 and transduction without myc‐p16 INK4a as a control.

Techniques: MANN-WHITNEY, Transduction, Over Expression

Journal: Aging Cell

Article Title: FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16 INK4a increase

doi: 10.1111/acel.13226

Figure Lengend Snippet: Gene expression analyses in human NSCs. The mRNA levels are normalized to cells treated with nontargeting control (NTC) siRNAs (siRNA tests) or to C116 cells or cells without growth factor (GF) deprivation (other experiments). ns, not significant. (a) ETS2 mRNA levels are increased by FOXO3 reduction in HD NSCs subjected to GF deprivation with no effect detected in basal conditions nor in normal HTT cells (left panel: * p < .05). ETS2 mRNA levels are decreased in HD NSCs (middle panel: ** p < .01). GF deprivation does not change ETS2 mRNA levels in C116 and decreases ETS2 mRNA levels in HD NSCs (right panel: * p < .05). (b) p16 INK4a mRNA levels are decreased by ETS2 reduction in HD NSCs in basal conditions and in cells subjected to stress with no effect detected in normal HTT cells (left panel: * p < .05, ** p < .01). p16 INK4a mRNA levels are increased in HD NSCs (middle left panel: *** p < .001). GF deprivation does not change p16 INK4a mRNA levels in C116 NSCs and decrease p16 INK4a mRNA levels in HD NSCs (middle right panel: * p < .05). p16 INK4a mRNA levels tend to be increased by FOXO3 knockdown in HD NSCs subjected to GF deprivation (right panel: not significant with p = .0736). (c) ETS2 and p16 INK4a mRNA levels are decreased by overexpression of FOXO3, but not that of FOXO3‐TM, in human HD NSCs subjected to GF deprivation. The mRNA levels are normalized to cells treated with empty vector. * p < .05 and ** p < .01. (d) p16 INK4a mRNA levels are decreased by ETS1 reduction in C116 NSCs in basal conditions and in HD NSCs in both basal and stress conditions (upper panel: * p < .05, ** p < .01). ETS1 mRNA levels are unchanged in HD compared with C116 NSCs (lower left panel). GF deprivation does not change ETS1 mRNA levels in C116 NSCs and decreases ETS1 mRNA levels in HD NSCs (lower right panel: * p < .05). (d) Working model for effect of FOXO3 target reprogramming on the ETS2‐p16 INK4a pathway

Article Snippet: Lentivirus transduction was performed with myc‐p16 INK4a from Origene (RC220937L1V) using a multiplicity of infection (MOI) of 1 and transduction without myc‐p16 INK4a as a control.

Techniques: Gene Expression, Control, Knockdown, Over Expression, Plasmid Preparation

Journal: Aging Cell

Article Title: FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16 INK4a increase

doi: 10.1111/acel.13226

Figure Lengend Snippet: Human HD prepatterned NSCs show increase of p16 INK4a and of SA‐β‐gal activity. (a) p16 INK4a mRNA levels are increased in HD prepatterned NSCs. Data are mean ± SD (** p < .01), N = 3. (b) Representative images for modest p16 INK4a increase in HD NSCs. Scale bar in all panels: 100 µm. (c) Quantification of nuclear p16 INK4a pixel intensity for 532 C116‐NSCs and 1000 HD NSCs. Data are mean ± SD (** p < .01). (d) Representative images for increase of SA‐ß‐gal activity in HD NSCs. Scale bar in all panels: 200 µm. (e) Quantification of SA‐ß‐gal activity for 547 C116‐NSCs and 645 HD NSCs. Data are mean ± SD (**** p < .0001). (f) Frequency distribution of SA‐ß‐gal signals for data shown in panel (e)

Article Snippet: Lentivirus transduction was performed with myc‐p16 INK4a from Origene (RC220937L1V) using a multiplicity of infection (MOI) of 1 and transduction without myc‐p16 INK4a as a control.

Techniques: Activity Assay

Journal: Aging Cell

Article Title: FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16 INK4a increase

doi: 10.1111/acel.13226

Figure Lengend Snippet: p16 INK4a expression is elevated in human HD MSNs. (a) Representative images of human NSC‐derived MSNs using defined enhanced media (Synaptojuice medium). (b) RT‐PCR analysis of p16 INK4a , FOXO3, and Ryk in C116 and HD MSNs reveals modest increase of FOXO3 mRNA levels and robust increase of p16 INK4a and Ryk mRNA levels in HD MSNs. Data are mean ± SD (* p < .05, *** p < .001). N = 3. (c) Immunofluorescence analysis reveals dramatic increase of p16 INK4a in HD MSNs. Scale bar in all panels: 100 µm. (d) Quantification of nuclear p16 INK4a pixel intensity for N = 596 C116 NSCs and N = 609 HD NSCs. Data are mean ± SD (**** p < .0001). (e) Frequency distribution of nuclear p16 INK4a signals for data shown in Panel (d). (e) RT‐PCR analysis of CDKN2AIP, MMP3, SELL, IGFBP7, EST1, and EST2 show increased mRNA levels in HD MSNs compared with C116 MSNs. Data are mean ± SD (* p < .05, ** p < .01, *** p < .001). N = 3

Article Snippet: Lentivirus transduction was performed with myc‐p16 INK4a from Origene (RC220937L1V) using a multiplicity of infection (MOI) of 1 and transduction without myc‐p16 INK4a as a control.

Techniques: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence