Volume 7, Issue 8, Pages 1329-1349 (August 2014) A Novel Chloroplast-Localized Pentatricopeptide Repeat Protein Involved in Splicing Affects Chloroplast Development and Abiotic Stress Response in Rice  Junjie Tan, Zhenhua Tan, Fuqing Wu, Peike Sheng, Yueqin Heng, Xinhua Wang, Yulong Ren, Jiulin Wang, Xiuping Guo, Xin Zhang, Zhijun Cheng, Ling Jiang, Xuanming Liu, Haiyang Wang, Jianmin Wan  Molecular Plant  Volume 7, Issue 8, Pages 1329-1349 (August 2014) DOI: 10.1093/mp/ssu054 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Phenotypic Characteristics of the wsl Mutant. (A–E) Phenotypes of wild-type (left) and wsl mutant (right) seedlings at the one- (A), two- (B), three- (C), four- (D), and five- (E) leaf stages. The mutant developed leaves (especially the third leaf) with white stripes predominantly along the edges. The white box above the seedlings shows enlarged views of the regions indicated in wild-type and wsl leaves, respectively. Bars = 1mm. (F) The juvenile white-striped leaves never reverted to green, even in mature plants. (G) Phenotypes of WT (left) and wsl (right) plants at the heading stage. (H) Comparison of the WT and wsl seeds (de-hulled). (I–M) Transmission electron microscopic images of cells from wild-type, white, and green sectors of wsl mutant at the three-leaf stage. (I) Mesophyll cells in wild-type plants showed normal, well ordered chloroplasts. (J–L) Cells in white sectors of the mutants displayed some abnormalities, including vacuolated plastids and lack of organized thylakoid membranes (J), lack of most organelles (K), and abnormal cell morphology (L). (M) Chloroplasts from green sectors of wsl seedlings were indistinguishable from those of wild-type. Scale bar: 1 μm in (I, M), 500nm in (L), 2 μm in (J, K). Molecular Plant 2014 7, 1329-1349DOI: (10.1093/mp/ssu054) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 Enhanced ABA Sensitivity of the wsl Mutant. (A) Five-day-old wild-type and wsl seedlings on media supplemented with increasing concentrations of ABA. Shoot (B) and root (C) lengths of wild-type and wsl seedlings in (A). The error bar represents ±SE of approximately 30 plants. Asterisks indicate statistically significant differences compared with the control (Student's t-test: * P < 0.05; ** P < 0.01). (D–F) Germination rates of wild-type and wsl seeds in the absence (D), and presence of 2 μM (E) or 5 μM (F) of ABA. The germination was defined as the appearance of the white tip of the coleoptiles at least 5 mm long. Data represent mean ± SD from three independent biological replicates. (G) Environmental scanning electron microscope images defining three levels of stomatal opening. Bar = 5 μm. (H) The percentage of three levels of stomatal opening in WT and wsl mutant seedlings after treated by water (–) or 100 μM ABA (+) treatment for 6 h (n >100 stomata in each group). Molecular Plant 2014 7, 1329-1349DOI: (10.1093/mp/ssu054) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 Salt and Sugar Stress Sensitivities of WT and wsl at Germination. Germination frequencies of wild-type and wsl seeds under normal conditions (control) (A), 2% glucose (B), 6% glucose (C), 100mM NaCl (D), 150mM NaCl (E), and 200mM NaCl (F). The germination was defined as the appearance of the white tip of the coleoptiles at least 5 mm long. Data represent mean ± SD from three independent biological replicates each containing at least 30 seeds per treatment. Molecular Plant 2014 7, 1329-1349DOI: (10.1093/mp/ssu054) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 Molecular Cloning of the WSL Gene. (A) The WSL locus was initially localized to the LS01–LS11 region in the chromosome 1 (Chr1). (B) Fine mapping of WSL which was spanned by BAC clones OJ1014_G12, B1144D11, B1064G04, P0496H05, P0468H06, OSJNBb0049O23, and P0697C12. The number of recombinants identified from an F2 population of 3607 recessive phenotypes is shown below each marker. (C) The WSL locus was narrowed to a 34-kb region between markers LS041 and LS06. (D) The region contained five open reading frames (ORFs) and a 6-bp deletion (underlined) in ORF2, resulting in a loss of two amino acids. (E) Complementation testing by PCR. The forward primer was from the plant genome, the reverse primer from the vector. The expected size was 4140bp. M, marker; pWSL, complemented plants. (F) Complementation of the wsl mutant. Wild-type plants (left), wsl mutants (middle), and mutants transformed with pWSL vector (right) are shown. M, marker; pWSL, mutant plants transformed with the pWSL vector. Molecular Plant 2014 7, 1329-1349DOI: (10.1093/mp/ssu054) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 Sequence Analysis of WSL. (A) The wild-type WSL gene comprised 10 exons and nine introns. A 6-bp deletion in the wsl mutant was in the fourth exon. (B) The WSL protein has 14 PPR motifs. Chloroplast transit peptides (CTP) in the N-terminal and mutation in the 5th PPR motif. (C) Comparison of the PPR motifs of WSL. Amino acids fully or partially conserved are shaded black and gray, respectively. The black box represents the two deleted amino acids. (D) Alignment of amino acid sequences with the highest identity with the WSL protein. The Arabidopsis homolog is annotated as EMB3103. GenBank accession numbers for the other protein sequences are: Medicago truncatula (Medicago: XP_003593032); Populus trichocarpa (Populus: XP_002299667); Sorghum bicolor (Sorghum: XP_002455801; Brachypodium distachyon (Brachypodium: XP_003569217). Conserved amino acids are highlighted in shades of black and the two deleted amino acids indicated by black boxes are highly conserved in these species. PPR repeats are indicated below the sequences by double-headed arrows. Molecular Plant 2014 7, 1329-1349DOI: (10.1093/mp/ssu054) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 Subcellular Localization and Expression Analysis of WSL. (A–D) Subcellular localization of WSL. Fluorescence signals were detected by confocal microscopy. (A) Green fluorescence from WSL–GFP fusion protein. (B) Red fluorescence of chloroplast auto-fluorescence. (C) Bright-field image under transmitted light. (D) Merged image of (A), (B), and (C). Bars in (A–D) represent 5 μm. (E) Wild-type plants (93–11) at the five-leaf and maturity stages were used for qRT–PCR. L2–L5 represent the second to fifth leaves. S, stem; R, root; ML, mature leaf; P, panicle. (F) Transcription level of WSL in in various organs (means ± SD, n = 3). (G) Expression analysis of genes involved in chlorophyll biosynthesis, chloroplast development, or photosynthesis by qRT–PCR. The RNA was isolated from wild-type and white sectors of wsl of 1-week-old seedling leaves. Data represent mean ± SD from three independent biological replicates, and an asterisk indicates a significant difference (Student's t-test,* P < 0.05; ** P < 0.01). Molecular Plant 2014 7, 1329-1349DOI: (10.1093/mp/ssu054) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 7 Severe Reductions of Plastid rRNAs and Photosynthetic Proteins in wsl Mutants. (A) One-week-old seedlings grown in high light density were prepared for Northern and Western blotting. (B) RNA gel blot hybridizations showing plastid rRNA defects in wsl mutants. About 5 μg of total leaf RNA from (A) were analyzed by hybridization with probes for plastid 16S and23S rRNA. The same filters detected with methylene blue are shown at the bottom: bands corresponding to cytosolic rRNAs (25S and 18S) and plastid rRNAs (16S and 23S*) are marked. 23S* is a breakdown product of plastid 23S rRNA. (C) Immunoblot analysis of photosynthetic protein abundance. Total leaf proteins were analyzed by probing immunoblots with antiserum to representative subunits of photosystem I (PsaA and PsaB), photosystem II (D1 and D2), RopB and ATP synthase (AtpB). The same filter was stained with Ponceau S to visualize total proteins (bottom) and the large subunit of Rubisco (RbcL) is indicated on the stained gel. The RNA used for Northern analysis and the protein used for Western analysis were isolated from wild-type and white sectors of wsl mutant. Molecular Plant 2014 7, 1329-1349DOI: (10.1093/mp/ssu054) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 8 Splicing Analysis of All Rice Chloroplast Transcripts with Introns in Wild-Type and White Sectors of wsl. The genes of transcripts are labeled at the left. Spliced (S) and unspliced (U) transcripts are indicated on the right. RT–PCR was performed with the RNA from leaves of 1-week-old seedlings. 23rRNA was used as a reference. Molecular Plant 2014 7, 1329-1349DOI: (10.1093/mp/ssu054) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 9 rpl2 Shows a Splicing Defect in the wsl Mutant. (A) Structure of the rpl2 gene. The primers used in (B) and the probes used in (C). (B) Amplification of rpl2 transcripts from WT, wsl, and complemented wsl plants. The expected size of the product from spliced transcripts was 822bp whereas that from unspliced transcripts was 1485bp. Molecular weight markers (M) are shown on the right, and the amplification product from genomic DNA (gDNA) is given in the sixth panel, the right side of which shows a lack of amplification products from the RNA templates used for reverse transcription, indicating that the 1485-bp product from the cDNA sample is from unspliced transcript, and is not contaminated by genomic DNA; 0, control with no template. (C) RNA gel blot assay of rpl2 splicing. Bands corresponding to mature spliced RNAs (s) and excised intron (i) are marked. The arrow indicates the rpl2 pre-mRNA that accumulates in the wsl mutant. The same blots stained with methylene blue to show the rRNAs as a loading control are shown below. (D) Comparison of transcript abundance of rpl2 in wild-type (Wt) and wsl mutant by qRT–PCR. (E) Immunoblot was probed in wild-type and mutant with an RPL2-specific antiserum. The same filter was stained with Ponceau S to visualize total proteins (bottom) and the large subunit of Rubisco (RbcL) is indicated on the stained gel. RNA used for RT–PCR and Northern analysis was isolated from wild-type and white sectors of wsl of the 1-week-old seedling leaves. Molecular Plant 2014 7, 1329-1349DOI: (10.1093/mp/ssu054) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 10 wsl Accumulates Additional ROS. (A) DAB staining of the leaves from WT or wsl mutant. (B) Quantitative measurement of H2O2 in 5-day-old seedlings of WT and wsl mutant (n = 3 repeats). (C) Typan blue staining of leaves from WT and wsl. (D–M) Relative expression levels of genes involved in subcellular ROS detoxification including alternative oxidases (AOXs) (D–F), ascorbate peroxidase (APXs) (G, H), superoxide dismutases (SODs) (I, J), and catalases (CATs) (K–M). RNA was extracted from 5-day-old seedlings without or with 2 μM ABA treatment. Data represent mean ± SD from three independent biological replicates, and an asterisk indicates a significant difference between WT versus wsl under the same conditions (* P < 0.05; ** P < 0.01). Molecular Plant 2014 7, 1329-1349DOI: (10.1093/mp/ssu054) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions