Volume 10, Issue 1, Pages 86-98 (January 2017) The RNA Editing Factor WSP1 Is Essential for Chloroplast Development in Rice  Zhiguo Zhang, Xuean Cui, Yanwei Wang, Jinxia Wu, Xiaofeng Gu, Tiegang Lu  Molecular Plant  Volume 10, Issue 1, Pages 86-98 (January 2017) DOI: 10.1016/j.molp.2016.08.009 Copyright © 2017 The Author Terms and Conditions

Figure 1 Phenotypic Comparison of the Wild-type and wsp1 Mutant Plants. (A) A comparison of leaves from wsp1 mutant and wild-type (WT) plants at the third-leaf stage. The box shows magnified views of the areas in the red boxes. Scale bar, 1 cm. (B) A comparison of leaves from wsp1 mutant and wild-type plants at the mature stage. Scale bar, 1 cm. (C) A comparison of stature between wsp1 and wild-type plants at the mature stage. Scale bar, 5 cm. (D) A comparison of panicles from wsp1 mutant and wild-type plants at the mature stage. Scale bar, 1 cm. Molecular Plant 2017 10, 86-98DOI: (10.1016/j.molp.2016.08.009) Copyright © 2017 The Author Terms and Conditions

Figure 2 Transmission Electron Microscopic Images of Chloroplasts from Third-Leaf Stage Plants. (A) Wild-type mesophyll cells. (B) Mesophyll cells from wsp1 plants. (C) Wild-type chloroplasts. (D and E) Chloroplasts from wsp1 plants. The chloroplasts of the wild-type plants had abundant, well-ordered membrane stacks, whereas the chloroplasts from the white portions of the wsp1 plants had almost no normal stacked membranous structures or grana (D). The chloroplasts from green portions of the wsp1 plants had normal stacked membranous structures and grana (E). Scale bars represent 2 μm in (A) and (B), 0.5 μm in (C), 1 μm in (D)–(E). Molecular Plant 2017 10, 86-98DOI: (10.1016/j.molp.2016.08.009) Copyright © 2017 The Author Terms and Conditions

Figure 3 Map-Based Cloning of WSP1. (A) The WSP1 locus was mapped to a 2.0-cM interval on chromosome 4L between the simple sequence repeat markers Indel1-1 and Indel1-2. (B) The location of the WSP1 locus was narrowed down to a 30-kb region between InDel markers 3017 and 3020 on BAC clone OSJNBa0041A02 using 520 F2 homozygous mutant plants. (C) Four open reading frames were predicted in the mapped region. The structure of WSP1 (ORF3). ATG and TAA are start and stop codons, respectively. Black boxes indicate exons; lines between the black boxes indicate introns. A single nucleotide change resulted in a missense mutation. (D and E) A comparison of the mutated site in Os04g51280 between wild-type (D) and wsp1 (E). The red arrows point to the normal site(T) in wild type in Figure 3D and mutated site(A) in wsp1 in Figure 3E. Molecular Plant 2017 10, 86-98DOI: (10.1016/j.molp.2016.08.009) Copyright © 2017 The Author Terms and Conditions

Figure 4 Functional Complementation at the WSP1 Locus. (A) The expression vector constructed using BAC clone OSJNBa0041A02. LB and RB denote the left border and right border. (B) The phenotypes of wild-type (WT), wsp1, and complemented T0 plants (cp) at the heading stage grown under Beijing field conditions. Scale bar, 5 cm. (C) The complemented T0 plants showed two peaks at the mutated site. The double peak was shown at the mutation site (red arrow), indicating the presence of normal transcripts of WSP1. (D and E) The complemented plants showed a restored panicle color and leaf color. Scale bar, 1 cm. (F) A dCAPS marker was used to detect the complemented lines. Molecular Plant 2017 10, 86-98DOI: (10.1016/j.molp.2016.08.009) Copyright © 2017 The Author Terms and Conditions

Figure 5 WSP1 Is Required for RNA Editing at Multiple Sites. (A) Three plastid genes and the corresponding five editing sites were not normally edited in wsp1 plants. The black boxes indicate RNA editing site(T) in wild type and non-editing site(C) in wsp1 in different plastid genes. (B) The proteins were hybridized with antibodies against ndhD, RpoB, and RPS14, respectively. Hsp90 was used as an internal control.Wt, wild-type. Molecular Plant 2017 10, 86-98DOI: (10.1016/j.molp.2016.08.009) Copyright © 2017 The Author Terms and Conditions

Figure 6 Analyses of rRNA and Ribosomal Proteins from the Wild-type and wsp1 Plants. (A–D) rRNA analysis using an Agilent 2100. RNA was isolated from 10-day-old wild-type seedlings (A), wsp1 seedlings (B), wsp1 panicles (C), and wild-type roots as a control (D). FU, fluorescence units. (E) Western blot analysis of chloroplast ribosomal proteins. Proteins were extracted from 10-day-old seedlings. The proteins were hybridized with antibodies against RPL2, RPS3, and RPS18, respectively. Hsp90 was used as an internal control. WT, wild-type. Molecular Plant 2017 10, 86-98DOI: (10.1016/j.molp.2016.08.009) Copyright © 2017 The Author Terms and Conditions

Figure 7 The ndhA Gene Displays a Splicing Defect in wsp1. (A) Structure of the ndhA gene with the two exons shown in gray boxes and the primers used in (B). (B) RT–PCR analysis of the ndhA transcripts from wild-type (Wt), wsp1, and cp plants. The product from the spliced transcripts had an expected size of 1100 bp, whereas that from the unspliced transcripts was 1956 bp. Molecular weight markers (M) are shown on the left; the product amplified from total DNA (gDNA) is shown in the center. The right side of the panel shows a lack of amplification by the RNA templates used for reverse transcription, indicating that the 1956-bp product from the cDNA sample resulted from an unspliced transcript and not from contaminating genomic DNA. (C) A qRT–PCR-based analysis of ndhA expression. (D) Total leaf proteins were analyzed by probing immunoblots with ndhA antiserum. The same amount was immunoblotted with Hsp90 antibodies (bottom). WT, wild-type. Molecular Plant 2017 10, 86-98DOI: (10.1016/j.molp.2016.08.009) Copyright © 2017 The Author Terms and Conditions

Figure 8 A Schematic of the Hypothesis Illustrating the Possible Mechanism Underlying the Variegated Phenotype of wsp1. The main characteristic of the wsp1 mutant is its deficiency in RNA editing, and the remaining defects (e.g., chlorophyll content, gene expression changes, ribosome biogenesis, RNA splicing) are secondary. Because the plastid genes rpoB and rps14 are not normally edited in the wsp1 mutant, two levels are capable of regulating chloroplast development, transcription, and translation. Transcription is hampered because PEP complexes are impaired in the wsp1 mutant. Translation does not normally proceed due to defects in the ribosome complex, including decreases in the ribosome protein level and the loss of rRNA in the wsp1 mutant. The PPR? indicates that there may be an unidentified PPR family member directly invovling into ndhA splicing. Molecular Plant 2017 10, 86-98DOI: (10.1016/j.molp.2016.08.009) Copyright © 2017 The Author Terms and Conditions