1. The Integrity of the Plant Golgi Apparatus Depends on Cell Growth-Controlled Activity of GNL1 
Wenyan Du, Kentaro Tamura, Giovanni Stefano, Federica Brandizzi 
Molecular Plant 
Volume 6, Issue 3, Pages (May 2013)
DOI: /mp/sss124
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
The pao Mutant Shows Abnormal Distribution of the Golgi Marker ST–GFP to a Meshwork of Circular Structures and Is Linked to a Novel Loss-of-Function Allele of GNL1.
(A) Confocal microscope image of epidermal cells of 3.5-DAG Arabidopsis cotyledons showing that, compared with the distribution of ST–GFP to Golgi stacks in non-mutagenized cells (Col-0-ST–GFP), the marker is partially distributed to circular structures, resembling bubbles in the pao mutant (pao/ST–GFP). Arrowheads indicate Golgi stacks and arrows indicate the circular structures, respectively. Scale bar = 5 µm.
(B) Schematic representation of GNL1 gene and indication of the position of the pao mutation (3405 bp) at mRNA level. Lower section shows the corresponding domain structure of GNL1. Exons are represented by black boxes and intron by a line.
(C) Confocal image of an epidermal cell of 3.5-DAG Arabidopsis cotyledons of either pao/ST–GFP expressing GNL1, which are complemented, or pao/ST–GFP x ermo1-2 F1 generation, which are not complemented. These data show that the pao phenotype is allelic to GNL1 and that pao is a loss-of-function allele. Scale bars = 5 µm.
Molecular Plant 2013 6, DOI: ( /mp/sss124)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

3. Figure 2
The Network Delineated by ST–GFP in the pao Mutant Corresponds to a Modified ER, which Accumulates Cargo for the Lumen of the Vacuole as well as for the Apoplast.Confocal microscopy of epidermal cells of 3.5-DAG Arabidopsis cotyledons of either the non-mutagenized Col-0 background (control) or pao expressing ER-YK ((A), ER), RFP-AFVY ((B), vacuole lumen), secRFP ((C), apoplast), or YFP-ABD2 ((D), actin). Co-localization analyses with these markers show that the circular structures visible in the pao mutant correspond to a compromised ER network in which vacuolar and apoplastic markers are partially retained. Such defects are independent of the actin cytoskeleton, since no appreciable differences were noted in the organization of the microfilaments in the pao mutant compared to wild-type. Stars indicate the distribution of ER-YK in the ER network, RFP-AFVY in the lumen of the vacuole, secRFP in the apoplast, and YFP-ABD2 at the actin, respectively. Insets: magnified regions of main images. Scale bars = 5 µm.
Molecular Plant 2013 6, DOI: ( /mp/sss124)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

4. Figure 3 GNL1 Confers BFA Resistance in Early Stages of Cell Growth.
(A) Schematic representation of stages of cotyledon development upon seed vernalization and germination. 3.5, 8.5 and 12.5 DAG correspond to the days that the sampling was performed in this work.
(B) Confocal images of 3.5, 8.5, and 12.5-DAG cotyledon epidermal cells of wild-type and pao mutant showing that, at 3.5 DAG, wild-type cotyledons are resistant to BFA but that, at 8.5 and mainly 12.5 DAG, they become sensitive. Conversely, pao cells, which lack a functional GNL1, are sensitive to BFA throughout cell growth. Treatments were performed for 1 h in either DMSO (mock: a, b) or BFA (50 µg ml–1: c, d; 100 µg ml–1: e, f). Scale bar in 3b is for columns a and b. Scale bar in 3f is for columns c, d, e, and f. Scale bars = 5 µm.
Molecular Plant 2013 6, DOI: ( /mp/sss124)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
With the Exception of the ER, the pao Mutant Phenotype of the Endomembranes Disappears as Cells Cease to Grow.Images of cotyledon epidermal cells of 3.5-DAG wild-type (WT row), as well as of 3.5, 8.5, and 12.5-DAG pao mutant (3.5, 8.5, and 12.5-DAG rows, respectively) expressing ST–GFP (a, Golgi), ER-YK (b, ER), secRFP (c, apoplast), and RFP-AFVY (d, vacuole lumen). Note that, differently from the ER marker, the pao phenotype is alleviated at 8.5 DAG and that it disappears at 12.5 DAG for the apoplast and vacuole markers. Arrowheads indicate Golgi stacks and arrows indicate circular structures. Stars in c and d indicate apoplast and vacuole, respectively. Scale bar = 5 µm.
Molecular Plant 2013 6, DOI: ( /mp/sss124)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

6. Figure 5 GNL1 Decreases in Abundance during Cell Growth.
(A) Quantitative RT–PCR analyses of Col-0/ST–GFP cotyledons show that the transcript levels of GNL1 (A) decrease from 3.5 through 8.5 and 12.5 DAG.
(B) Confocal images of cotyledon epidermal cells expressing Col-0/GNL1::GNL1–YFP at 3.5, 8.5, and 12.5 DAG show a marked decrease in the fluorescence over time.
(C) Quantitation of fluorescence signal on punctae labeled by GNL1::GNL1–YFP (normalization to 3.5 DAG as 100% intensity). For this assay, we measured the fluorescence intensity on the Golgi where GNL1 has been shown to accumulate (Richter et al., 2007; Teh and Moore, 2007) using identical non-saturating confocal imaging settings throughout the time course (i.e. magnification, laser intensity, pinhole aperture, channel gain). We established that the fluorescence of GNL1–YFP from 3.5 DAG decreased at 8.5 and 12.5 DAG. Scale bar = 5 µm.
Molecular Plant 2013 6, DOI: ( /mp/sss124)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions