Volume 4, Issue 2, Pages 229-240 (March 2011) SUPPRESSOR OF VARIEGATION4, a New var2 Suppressor Locus, Encodes a Pioneer Protein that Is Required for Chloroplast Biogenesis  Yu Fei , Park Sung-Soon , Liu Xiayan , Foudree Andrew , Fu Aigen , Powikrowska Marta , Khrouchtchova Anastassia , Jensen Poul Erik , Kriger Jillian N. , Gray Gordon R. , Rodermel Steven R.   Molecular Plant  Volume 4, Issue 2, Pages 229-240 (March 2011) DOI: 10.1093/mp/ssq074 Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 1 Growth Phenotype of Wild-Type Columbia, var2-5 and the var2-5 Suppressor Strain, ems2505 The plants were maintained for 2 weeks under the same conditions (100 μmol m−2 s−1 continuous light at 22°C). Molecular Plant 2011 4, 229-240DOI: (10.1093/mp/ssq074) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 2 Map-Based Cloning of the Suppressor Gene SVR4 (A) SSLP markers were used to map the suppressor gene between ciw7 and nga1107 on chromosome 4. The gene was fine-mapped to a ∼120-kb region between markers DVcM13.0284 and DVcM13.1505 (Supplemental Table 1). This region is covered by three BAC clones: F20O9, T5F17, and F16A16. (B) The suppressor gene (At4g28590) is on BAC clone T5F17. It was designated SVR4. Our cDNA amplifications confirmed the annotation of At4g28590, showing that the gene contains four exons (boxes) and encodes a 331-amino acid protein. The gene also contains a predicted chloroplast targeting sequence (Met1-Arg92; gray box). The svr4-1 mutation is in the 3rd exon and changes Arg291 to Trp291 (R291W). A T-DNA mutant was acquired from the SALK collection (SALK_075057) and its insertional locus is shown in intron 2. Molecular Plant 2011 4, 229-240DOI: (10.1093/mp/ssq074) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 3 SVR4 and SVR4-Like Genes in Plants. (A) Phylogenetic relatedness of SVR4 (At4g28590) and SVR4-like genes (At2g31840 in Arabidopsis) in plants. Full-length protein sequences were obtained from NCBI. The phylogenetic tree was constructed using MEGA4 software (Tamura et al., 2007). (B) Gene structures of SVR4 and SVR4-like genes. Boxes denote exons and lines indicate introns. Untranslated regions (UTRs) of each gene are shown as boxes with hatched lines. The number of nucleotides in each exon is shown. Molecular Plant 2011 4, 229-240DOI: (10.1093/mp/ssq074) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 4 Complementation of ems2505. (A) Two-week-old wild-type, var2-5, ems2505, and ems2505 P35S:SVR4 seedlings. (B) RNA gel blot analysis of SVR4 mRNAs from the same plants as in (A). The blot was probed with 32P-labeled SVR4 cDNAs. The ethidium bromide-stained RNA gel is shown as a loading control. Molecular Plant 2011 4, 229-240DOI: (10.1093/mp/ssq074) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 5 Sub-Cellular Localization of SVR4. Protoplasts from wild-type Arabidopsis were transiently transformed with a control GFP vector (designated P35S:GFP) (A–C) or with a SVR4–GFP vector (designated P35S:SVR4-GFP) (D–F). Fluorescence was observed by confocal microscopy of single protoplasts: green fluorescence of GFP (A, D); red chlorophyll autofluorescence (B, E); merged images (C, F). Molecular Plant 2011 4, 229-240DOI: (10.1093/mp/ssq074) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 6 Accumulation of Representative Thylakoid Membrane Proteins in Wild-Type and svr4-1 Plants. Thylakoid membranes were isolated from 20-day-old seedlings (wild-type, svr4-1, var2-5, and ems2505), and proteins were separated by 12.5% SDS–PAGE. Loadings were on an equal chlorophyll basis. The proteins were transferred from the gel to a nitrocellulose membrane, and the filter was immunoblotted with antibodies to VAR2, AtFtsH1, D1, PsbS, PsaN, PsaF, ATPa, Rieske Fe/S protein, and Lhcb2. Molecular Plant 2011 4, 229-240DOI: (10.1093/mp/ssq074) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 7 Non-Photochemical Quenching (NPQ) in Leaves of Wild-Type (•) and svr4-1 (○). Rapid induction and dark relaxation (A), as well as steady-state (B) determinations are shown. In (A), white bar indicates time period of induction and black bar indicates relaxation time period. An actinic irradiance of 2200 μmol m−2 s−1 was used for induction. When not present, error bars are smaller than symbol size. Values represent the means ± SD of three independent experiments. Molecular Plant 2011 4, 229-240DOI: (10.1093/mp/ssq074) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 8 Steady-State Light Response Curves in Leaves of Wild-Type (•) and svr4-1 (○). Fluorescence parameters include 1-qp (A), Fv’/Fm’ (B), ΦPSII (C), and ETR (D). When not present, error bars are smaller than symbol size. Values represent the means ± SD of three independent experiments. Molecular Plant 2011 4, 229-240DOI: (10.1093/mp/ssq074) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 9 SVR4 Is Essential for Seedling Development. (A) Two-week-old wild-type, svr4-2/svr4-2, and svr4-2 P35S:SVR4 seedlings. (B) Three-week-old wild-type, svr4-1/svr4-1, and svr4-1/svr4-2 seedlings. (C) The accumulation of thylakoid membrane proteins in svr4-2. Proteins were loaded on a fresh weight basis. Western blots were probed with the same set of antibodies as in Figure 6. Molecular Plant 2011 4, 229-240DOI: (10.1093/mp/ssq074) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 10 Chloroplast Ultrastructure in Wild-Type and svr4-2. Transmission electron microscopy (TEM) was performed using cotyledons from 10-day-old wild-type (A) and svr4-2 (B, C). Numerous plastoglobuli (PG) and small vacuoles (V) are seen in the svr4-2 seedlings. Bar = 0.5 μm. Molecular Plant 2011 4, 229-240DOI: (10.1093/mp/ssq074) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 11 SVR4 Transcript Expression. (A) Expression ratio of SVR4 (At4g28590; log based 2) transcripts under different hormone, light and abiotic stress treatments. Details of treatments are described at the AtGene Express webpage (www.uni-tuebingen.de/plantphys/AFGN/). A stringent cut-off is 1.5 (as in Rosso et al., 2006). (B) Differential expression of SVR4 transcripts in different tissues. The expression level is based on MAS 5.0 scaling by NASC (see Methods) to give relative expression levels; 100 units is the genomic average. * indicates the tissues that showed at least three times more transcription of SVR4 than the genome mean value. Molecular Plant 2011 4, 229-240DOI: (10.1093/mp/ssq074) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions