Volume 9, Issue 7, Pages 1082-1084 (July 2016) A Mitochondria-Targeted PPR Protein Restores pol Cytoplasmic Male Sterility by Reducing orf224 Transcript Levels in Oilseed Rape  Zhi Liu, Zonghui Yang, Xiang Wang, Kaidi Li, Hong An, Jie Liu, Guangsheng Yang, Tingdong Fu, Bin Yi, Dengfeng Hong  Molecular Plant  Volume 9, Issue 7, Pages 1082-1084 (July 2016) DOI: 10.1016/j.molp.2016.04.004 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Cloning and Functional Analysis of the Restorer Gene Rfp for pol CMS in Oilseed Rape. (A) Morphology of flowers, anthers, and pollen grains of the pol CMS plant and the hemizygous ORF2 transgenic plant. Partially dissected flowers were photographed with a digital camera. Anthers were photographed under a scanning electron microscope. Pollen grains were stained with 1% aceto-carmine staining solution. Scale bars, 50 μm. (B) qRT–PCR analysis of Rfp expression patterns in selected tissues. Sd, seeds; R, root; St, stem, L, leaves; B, buds; F, opening flowers; P, pods. The expression levels were normalized to BnActin. The values are presented as the means ± SD (n = 3 biological replicates). (C) The expression patterns of Rfp by RNA in situ hybridization in the anthers of B409 at different developmental stages. The Rfp sense-strand probe as a negative control produced no hybridization signal at the tetrad stage. E, epidermis; En, endothecium; MC, meiotic cell; ML, middle layer; MMC, microspore mother cells; MSp, microspores; PG, pollen grains; T, tapetum; Tds, tetrads. Scale bars, 50 μm. (D) Subcellular localization of full-length RFP fused with green fluorescent protein (GFP). The protoplast showed a green fluorescent signal at 488 nm; the same protoplast showed a red fluorescent signal (stained by MitoTracker Red CMX-Ros) at 580 nm. A bright-field image and the merged image are shown at the bottom. Scale bars, 10 μm. (E) orf224 expression in flower buds of 1141B, 1141A, B409 and three Rfp transgenic T1 plants analyzed by qPCR. BnActin was used as an internal control. Data are represented as means ± SD (n = 3 biological replicates). (F) RNA gel blot analysis of transcripts from maintainer, CMS, fertility-restored line; and transgenic-restored plants using orf224 as a probe and 18srRNA probe as a control. Lane 1, maintainer line; lane 2, CMS line; lane 3, nuclear fertility-restored line; lane 4, transgenic-restored lines; M, molecular markers. Molecular Plant 2016 9, 1082-1084DOI: (10.1016/j.molp.2016.04.004) Copyright © 2016 The Authors Terms and Conditions