Volume 4, Issue 5, Pages 832-844 (September 2011) CGR3: A Golgi-Localized Protein Influencing Homogalacturonan Methylesterification  Held Michael A. , Be Evan , Zemelis Starla , Withers Saunia , Wilkerson Curtis , Brandizzi Federica   Molecular Plant  Volume 4, Issue 5, Pages 832-844 (September 2011) DOI: 10.1093/mp/ssr012 Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 1 CGR3–cCFP Partially Co-Localize with the ManII–YFP Golgi Marker. Live cell confocal laser scanning microscopy of Arabidopsis leaf epidermal cells expressing CGR3–cCFP and ManII–YFP shows that CGR3–cCFP (pseudocolored green) is localized to the Golgi stacks (arrowheads) labeled by the Golgi marker, ManII–YFP (pseudocolored red), as indicated by arrows in the merged image. Scale bar = 10 μm. Molecular Plant 2011 4, 832-844DOI: (10.1093/mp/ssr012) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 2 CGR3 (At5g65810) Encodes a Putative MT-Domain-Containing Type II Membrane Protein. (A) Membrane topology and transmembrane domain prediction of CGR3 was performed using the TMHMM software from (www.cbs.dtu.dk/services/TMHMM-2.0/). Note that ‘outside’ delineates a predicted luminal orientation of the protein domain, while ‘inside’ refers to a predicted cytosolic portion of the protein. (B) Proteins from 7-day-old Arabidopsis seedlings expressing either CGR3–cCFP or ST–GFP were separated into soluble (S) and insoluble pellet (P) fractions then detected by Western blot with an anti-GFP antibody. Asterisks indicate the predicted molecular sizes of CGR3–cCFP (55.3 kDa) and ST–GFP (33 kDa). Wt indicates extracts from untransformed Col-0 seedlings. Protein molecular size markers (kDa) are shown to the right of the blot. Molecular Plant 2011 4, 832-844DOI: (10.1093/mp/ssr012) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 3 Identification of a T-DNA Knock-Out Mutant for CGR3. (A) Protein coding gene model of CGR3 as predicted by TAIR (version 9). Exons are represented by solid black bars. The 5' and 3' untranslated regions are represented by solid gray bars, and introns are represented by black lines. The T-DNA insertion site for cgr3-1 is marked by a black triangle and is located 2 bp upstream from the start codon (+1), according to TAIR v.9. (B) RT–PCR was performed on cDNA synthesized from three individual WT Col-0 plants and three individual homozygous cgr3-1 plants using primers for CGR3 expression (cgr3). Amplification of ubiquitin 10 (Ubi) was performed as a loading control. Molecular Plant 2011 4, 832-844DOI: (10.1093/mp/ssr012) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 4 Changes in the Expression of CGR3 Induce Morphological Changes as Well as Changes in the Methylesterification of Homogalacturonans. (A) Photos of 25-day-old Arabidopsis seedlings. Elongated petioles were observed for plants expressing CGR3–cCFP. Scale bar = 1 cm. (B) The lengths of similar age petioles (indicated by arrows in (A)) were measured from 25-day-old plants. Shown here are the averages of 20 measurements each for wild-type Col-0, CGR3–cCFP, and cgr3-1 mutant plants ± SD. The asterisk indicates p < 0.05 by paired Student's t-test. (C) Transverse sections of petioles (22 d) collected from wild-type Col-0 (WT), cgr3-1, 35S::CGR3–cCFP, and qua2-1 (Mouille et al., 2007) were probed for pectin epitopes using the JIM5, LM19, JIM7, and LM20 antibodies. Control sections lacking the addition of primary antibody (–CTRL) were examined to estimate background fluorescence. All immunofluorescence sections are in the same scale (bar = 50 μm). Molecular Plant 2011 4, 832-844DOI: (10.1093/mp/ssr012) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 5 Cell Wall Monosaccharide Analyses of cgr3-1 and CGR3–cCGP Are Similar to Wild-Type. (A, B) Alcohol-insoluble residues (AIR) were prepared from etiolated hypocotyls (7 d) and analyzed for neutral sugar compositions as alditol acetate derivatives (Cavalier et al., 2008). Neutral sugar compositions were expressed in terms of mol percentages (A) and in terms of weight recoveries (μg of monosaccharide per mg AIR) (B). Data represent the mean values of triplicate technical measurements ± SD and asterisks indicate p < 0.05 by paired Student's t-test. (C) Uronic acids from the AIR were measured using a colorimetric method (Filisetti-Cozzi and Carpita, 1991) and are expressed as the total nmol of uronic acid per mg AIR. qua2-1 samples were included as a control for the assay (Mouille et al., 2007). These data represent the average of three independent experiments ± SD and asterisks indicate p < 0.05 by paired Student's t-test. Molecular Plant 2011 4, 832-844DOI: (10.1093/mp/ssr012) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 6 Biochemical Estimation of the Degree of Esterification (DE). Alcohol-insoluble residues prepared from wild-type Col-0, cgr3-1, CGR3–cCFP, and qua2-1 etiolated hypocotyls were analyzed for methylesterification by saponification and detection of liberated methanol (Wood and Siddiqui, 1971). Data represent the average of three technical replicates ± SD and are presented as the molar ratio of methanol to uronic acid content. Asterisks indicate p < 0.05 by paired Student's t-test. Molecular Plant 2011 4, 832-844DOI: (10.1093/mp/ssr012) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions