Volume 4, Issue 6, Pages (November 2011)

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Volume 4, Issue 6, Pages 1062-1073 (November 2011) Localization of the Arabidopsis Senescence- and Cell Death-Associated BFN1 Nuclease: From the ER to Fragmented Nuclei Farage-Barhom Sarit , Burd Shaul , Sonego Lilian , Mett Ana , Belausov Eduard , Gidoni David , Lers Amnon Molecular Plant Volume 4, Issue 6, Pages 1062-1073 (November 2011) DOI: 10.1093/mp/ssr045 Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 1 Localization of BFN1–GFP in Protoplasts during Culture Senescence. (A) Protoplasts were observed 24, 48, and 72 h after co-transformation with vectors for expression of BFN1–GFP (green) and MBD5–RFP (red). Chloroplast autofluorescence is visible (blue). As a control, localization of GFP protein (green) in the protoplast is shown (CON). (B) Percentage of viable protoplasts at different time points following transformation. (C) Agarose gel analysis of protoplasts genomic DNA at different times during protoplasts’ culture senescence. Numbers indicate hours after transformation; M, DNA marker. Molecular Plant 2011 4, 1062-1073DOI: (10.1093/mp/ssr045) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 2 Cellular Localization of BFN1–GFP in Leaves of Transgenic Arabidopsis Plants. Localization of BFN1–GFP (green) in cells of a young leaf (A) and in cells from different sections of a senescing leaf (black boxes) that are at different stages of senescence: early (B), intermediate (C), and late (D). Higher magnifications of individual cells from the corresponding areas of the senescing leaf are shown in (E–G): nuclei stained with PI are in red and chloroplast autofluorescence is in blue. Molecular Plant 2011 4, 1062-1073DOI: (10.1093/mp/ssr045) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 3 BFN1–GFP Co-Localization with Nucleic Acids in Cells at a Late Stage of Senescence. Transgenic Arabidopsis leaves expressing BFN1–GFP (green) at a very late stage of senescence were stained with PI (red) (A, B). VL, visible light. Chlorophyll autofluorescence is shown in blue. Molecular Plant 2011 4, 1062-1073DOI: (10.1093/mp/ssr045) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 4 Localization of BFN1–GFP in Developing Vascular Tissues of Young Cotyledons. Confocal microscopy of cotyledons from transgenic Arabidopsis expressing BFN1–GFP (green). Shown are cells in a region undergoing differentiation of vascular tissues. VL, visible light. Molecular Plant 2011 4, 1062-1073DOI: (10.1093/mp/ssr045) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 5 Localization of BFN1–GFP Expressed under the Control of the BFN1 Promoter. Confocal microscopy of leaves from transgenic Arabidopsis expressing BFN1–GFP (A–C) or the control GFP (D) under the regulation of the BFN1 promoter (green). Leaves were stained with PI (red) for nuclear localization. Leaves at different developmental stages were examined: young (A), early senescence (B), and late senescence (C, D). VL, visible light. Molecular Plant 2011 4, 1062-1073DOI: (10.1093/mp/ssr045) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 6 Localization of BFN1–GFP Relative to Localization of LysoTracker and FM4-64 Markers. Confocal microscopy of leaves from transgenic Arabidopsis expressing BFN1–GFP (green). Cells from leaf tissues at different stages of senescence: early (A, D), intermediate (B), and late (C, E) were stained with FM4-64 (red) (A–C) or LysoTracker (red) (D, E). VL, visible light. Molecular Plant 2011 4, 1062-1073DOI: (10.1093/mp/ssr045) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 7 Co-Localization of the BFN1–GFP Protein with ER Marker. Confocal microscopy of protoplast cultures 24 (A), 48 (B), and 72 (C, D) hours after transformation with vectors expressing BFN1–GFP (green) and ER-rk (red) (A–C), or GFP and ER-rk as a control (D). VL, visible light. Molecular Plant 2011 4, 1062-1073DOI: (10.1093/mp/ssr045) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions