Volume 2, Issue 6, Pages 1211-1222 (November 2009) Arabidopsis FtsZ2-1 and FtsZ2-2 Are Functionally Redundant, But FtsZ-Based Plastid Division Is Not Essential for Chloroplast Partitioning or Plant Growth and Development  Schmitz Aaron J. , Glynn Jonathan M. , Olson Bradley J.S.C. , Stokes Kevin D. , Osteryoung Katherine W.   Molecular Plant  Volume 2, Issue 6, Pages 1211-1222 (November 2009) DOI: 10.1093/mp/ssp077 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 1 Comparison of the ftsZ T-DNA Insertion Mutants. (A) Positions of the previously identified ftsZ SALK T-DNA insertions (SALK_073878 (1–1 KO) (Yoder et al., 2007), SALK_134970 (2–1 KD), and SALK_050397 (2–2 KO) (McAndrew et al., 2008)) and the newly identified ftsZ2-1 GABI-Kat T-DNA insertion (596H04 (2–1 KO)). Exons are indicated by boxes and introns by lines between exons (Stokes and Osteryoung, 2003) (intron 1 for FtsZ2-1 and FtsZ2-2 is upstream of the start codon and is not shown). 25 codons are missing from exon 1 in the ftsZ2-1 GABI-Kat line (see Supplemental Figure 2A for more detail). (B) Immunoblot of the T-DNA lines from Figure 1A using antibodies specific for FtsZ1-1, FtsZ2-1, and FtsZ2-2 (α1–1, α2–1, and α2–2), respectively. WT Seg and 2–1 Het refer to WT and heterozygous segregants of the GABI-Kat T-DNA insertion. Equal amounts of chlorophyll (1 μg) were loaded and Ponceau S staining (P S) of Rubisco was performed to confirm equal loading (longer exposure is shown in Supplemental Figure 2B). (C) Bright-field images of mesophyll chloroplast phenotypes for WT, and the 1–1 KO, 2–2 KO, and 2–1 KD mutants (black labels) and Nomarski DIC images of the WT, 2–1 KO, and 2–1 KD (white labels). Bars = 10 μm. (D) Graph of chloroplast number relative to cell size. The best-fit lines have slopes of 0.0196 (R2  =  0.86), 0.0096 (R2  =  0.76), 0.0017 (R2  =  0.05), and 0.0009 (R2  =  0.01) for WT, 2–2 KO, 2–1 KD, and 1–1 KO, respectively. Molecular Plant 2009 2, 1211-1222DOI: (10.1093/mp/ssp077) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 2 The PFtsZ2-1::FtsZ2-2 Transgene Complements the 2–1 KO Phenotype. (A) Nomarski DIC images of mesophyll chloroplasts in WT, 2–1 KO, and 2–1 KO/PFtsZ2-1::FtsZ2-2 T1 lines with different chloroplast numbers (T1 Comp1 (WT chloroplast numbers), T1 Int1 (intermediate chloroplast numbers), and T1 Severe (greatly enlarged and often variable chloroplast sizes)). Bar  =  10 μm. (B) Immunoblot analysis of 2–1 KO/PFtsZ2-1::FtsZ2-2 T1 lines. T1 lines are labeled as complemented (C), intermediate (I), or severe (S) based on the size and number of mesophyll cell chloroplasts. Estimates of FtsZ2-2 protein levels relative to those in WT are shown. Equal amounts of chlorophyll (0.6 μg) were loaded except in WT lanes where 2.5-fold (2.5×) or 3.5-fold (3.5×) more chlorophyll was loaded. P S  =  Ponceau S staining of Rubisco. Molecular Plant 2009 2, 1211-1222DOI: (10.1093/mp/ssp077) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 3 The PFtsZ2-1::FtsZ2-1 Transgene Complements the 2–2 KO Phenotype. (A) Bright-field images of mesophyll chloroplast phenotypes for WT, 2–2 KO, and two 2–2 KO/PFtsZ2-1::FtsZ2-1 T1 lines with WT chloroplast numbers (T1 Comp1, T1 Comp2). Bar  =  10 μm. (B) Graph of chloroplast number relative to cell size. The best-fit lines have slopes of 0.0196 (R2  =  0.86), 0.0201 (R2  =  0.95), 0.0184 (R2  =  0.81), and 0.0096 (R2  =  0.76) for WT, T1 Comp2, T1 Comp1, and 2–2 KO, respectively. (C) Immunoblot analysis of T1 Comp1 (C1) and T1 Comp2 (C2) relative to WT. Estimates of FtsZ2-1 protein levels relative to those in WT are shown. Equal amounts of chlorophyll (0.8 μg) were loaded except in WT lanes, where two-fold (2×) or 3.5-fold (3.5×) more chlorophyll was loaded. P S  =  Ponceau S staining of Rubisco. Molecular Plant 2009 2, 1211-1222DOI: (10.1093/mp/ssp077) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 4 FtsZ2-2 Interacts with ARC6 in Yeast Two-Hybrid Analysis. Serial dilutions of yeast strains were tested for activation of the HIS reporter gene by plating on medium with (+HIS) or without (–HIS) histidine. The protein combinations tested are shown on the left (GAL4BD vs. GAL4AD vectors). ΔCT indicates a 16 amino acid C-terminal deletion of the specified FtsZ2 protein. Empty refers to empty GAL4BD or GAL4AD controls. + and – on the right indicate growth or no growth without histidine. Pictures were taken after 2 d of growth. Molecular Plant 2009 2, 1211-1222DOI: (10.1093/mp/ssp077) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 5 FtsZ1 and FtsZ2 Are Not Interchangeable for Chloroplast Division. (A) Immunoblot analysis was performed on 1–1 KO/PFtsZ2-1::FtsZ2-1 T1 individuals. Estimates of FtsZ2-1 protein levels relative to those in WT are shown. All transformants had severe (S) chloroplast division phenotypes. ^ indicates that the transformant has fewer chloroplasts per mesophyll cell than the 1–1 KO. Equal amounts of chlorophyll (0.65 μg) were loaded except in WT lanes, where 1.8-fold (1.8×) or 2.2-fold (2.2×) more chlorophyll was loaded. (B) Immunoblot analysis was performed on 2–1 KD/PFtsZ2-1::FtsZ1-1 T1 individuals. Estimates of FtsZ1-1 protein levels relative to those in WT are shown. All transformants had severe (S) chloroplast division phenotypes. ^ indicates that the transformant has fewer chloroplasts per mesophyll cell than the 2–1 KD. About 0.65 μg of chlorophyll was loaded per sample, except in WT lanes, where two-fold (2×) or 2.3-fold (2.3×) more chlorophyll was loaded. P S  =  Ponceau S staining of Rubisco. (C) Nomarski DIC images of mesophyll chloroplast phenotypes typical for 1–1 KO/PFtsZ2-1::FtsZ2-1 T1 lines. T1 lines S5 and S8 are the same individuals as in Figure 5A. Bar  =  10 μm. (D) Bright-field images of mesophyll chloroplast phenotypes typical for 2–1 KD/PFtsZ2-1::FtsZ1-1 T1 lines. T1 lines S5 and S7 are the same individuals as in Figure 5B. Bar  =  10 μm. Molecular Plant 2009 2, 1211-1222DOI: (10.1093/mp/ssp077) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 6 Identification of an ftsZ2 double KO. (A) Immunoblot analysis of 2–1 KO × 2–2 KO F2 individuals. Asterisks indicate lines lacking both FtsZ2-1 and FtsZ2-2. Extract from approximately 0.75 mg fresh weight of tissue was loaded per lane. P S  =  Ponceau S staining of Rubisco. (B) Nomarski DIC images of the mesophyll chloroplast division phenotype of ftsZ2 double KO (line A of Figure 6A) with WT, 2–1 KO, and 2–2 KO controls. Bar  =  10 μm. (C) The ftsZ2 double KO and the ftsZ triple KO have pale petioles. WT, other ftsZ mutant combinations, and arc6 are shown for comparison. Plants were photographed at 5.5 weeks post-germination. Molecular Plant 2009 2, 1211-1222DOI: (10.1093/mp/ssp077) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 7 Identification of an ftsZ triple KO. (A) Immunoblot analysis of ftsZ2 double KO × 1–1 KO/2–2 KO F2 individuals. Asterisks indicate the lines lacking detectable FtsZ1-1, FtsZ2-1, and FtsZ2-2 protein. Extract from approximately 1 mg fresh weight of tissue was loaded per lane. P S  =  Ponceau S staining of Rubisco. (B) Nomarski DIC images of the mesophyll chloroplast division phenotype of the ftsZ triple KO (line A of Figure 7A) with WT, 1–1KO, and ftsZ2 double KO controls. Bar  =  10 μm. Molecular Plant 2009 2, 1211-1222DOI: (10.1093/mp/ssp077) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions