Fig 1.
Morphological features of fleshy roots and flowers in the Ogura-CMS line and its maintainer fertile (MF) line of turnip.
(A) A MF plant and its fleshy roots at 110 days after germination. (B) An Ogura-CMS plant and its fleshy roots at 110 days after germination. (C) The length and diameter of fleshy roots at 110 days after germination. The values are the mean ± SD (standard deviation). Asterisks indicate statistical significance versus the length of MF fleshy root (**P<0.01); crosses indicate statistical significance versus the diameter of MF fleshy root (++P<0.01). Statistical significance is determined by a one-way ANOVA test. (D) A MF floret at anthesis stage with normal floral organs. (E) An Ogura-CMS floret at anthesis stage with short filaments and withered white anthers. Bars = 5 cm in (A, B), 2 mm in (D, E).
Fig 2.
Scanning electron microscopy observation of flower development in the Ogura-CMS line and its maintainer fertile (MF) line of turnip.
(A-D) Floral bud morphology in the MF line from the microspore mother cell stage to the mature pollen stage. (E) A MF dehiscent anther with normal oval pollen grains (inset shows the mature pollen grains). (F-I) Floral bud morphology in the Ogura-CMS line from the microspore mother cell stage to the mature pollen stage. (A, F) Microspore mother cell stage and tetrad stage. (B, G) Uninucleate stage. An Ogura-CMS floral bud exhibits the same morphology as a MF bud. (C, H) Bicellular stage. The withered anthers of the Ogura-CMS line are evident, compared with the lump anthers of the MF line. (D, I) Dehiscent stage. The MF anthers split open along the stomium, whereas the collapse of the Ogura-CMS anthers is evident. (J) An Ogura-CMS anther without any pollen grains. Bars = 1 mm in (A, B, E-H), 2 mm in (C, D, I), 500 μm in (J).
Fig 3.
Anther and microspore development in the Ogura-CMS line and its maintainer fertile (MF) line of turnip.
(A-E) Semi-thin sections of the MF anthers. (F-J) Semi-thin sections of the Ogura-CMS anthers. (A, F) Microspore mother cell stage. (B, G) Tetrad stage. The young microspores are surrounded by a callose wall, a tapetum, a middle layer, an endothecium, and an epidermis from the inside out at the tetrad stage. The tapetum in (G) swells at the center of the locule. (C, H) Uninucleate microspore stage. The middle layer persisted in (J). The aborted microspores indicated by arrowheads in (J) was surrounded by a swollen tapetal layer. (D, I) Bicellular stage. The collapse of anther locule is obvious with the aborted microspores indicated by arrowhead in (I). (E, J) Dehiscent stage. Endothecium layer is absent in the surrounding walls and remnants of the aborted microspores adhere to the inner face of the epidermis in (J). CL, collapsed locule; E, epidermis; En, endothecium; M, microspore; ML, middle layer; MMC, microspore mother cell; PG, pollen grain; RM, remnants of microspores; T, tapetum; Td, tetrads. Bars = 50 μm.
Fig 4.
Transmission electron microscopy observation of microspore development in the Ogura-CMS line and its maintainer fertile (MF) line of turnip.
(A-E) Images of microspore development in the MF line from the microspore mother cell stage to the mature pollen stage. (F-J) Images of microspore development in the Ogura-CMS line from the microspore mother cell stage to the mature pollen stage. (A, F) Microspore mother cell stage. (B, G) Tetrad stage, showing four young microspores surrounded by the callose wall. (C, H) Uninucleate microspore stage, showing the intine and the germinal apertures commenced in (C). (D, I) Bicellular stage, showing the degenerated microspores in (I). (E, J) Mature pollen stage, showing the mature pollen grain in the MF line (E) and the remnants of microspores in the Ogura-CMS line (J). (K-M) Magnified images of pollen wall in (C-E), showing the multilayered structure. (N, O) Magnified images of pollen wall in (H, I), showing the incomplete-developed exine layer and the absence of inine layer. Ba, baculum; CW, callose wall; Ex, exine; GA, germinal aperture; In, intine; M, microspore; MMC, microspore mother cell; Ne I, nexine I; Ne II, nexine II; PG, pollen grain; RM, remnants of microspores; Td, tetrads; Te, tectum; Tr, tryphine. Bars = 2 μm in (A-J), 0.2 μm in (K-O).
Fig 5.
Transmission electron microscopy observation of tapetum development in the Ogura-CMS line and its maintainer fertile (MF) line of turnip.
(A, F) Microspore mother cell stage. Micropores mother cells are surrounded by the tapetum, middle layer, endothecium, and epidermis from the inside out. (B, G) Tetrad stage, showing four distinctive surrounding walls and vacuolated tapetums. The tapetal cells in (G) swell to expand at the center of the locule, with larger vacuoles and a clearing cytoplasm. (C, H) Uninucleate microspore stage. Middle layer disappears and elaioplasts emerge in (C), whereas middle layer persists and tapetosomes were ubiquitous in (H). (D, I) Bicellular stage. Premature degradation of the tapetum occurs in (I), compared with integral tapetal cells with a large amount of elaioplasts in (D). (E, J) Mature pollen stage, showing the absence of the endothecium in (J). E, epidermis; En, endothecium; Ep, elaioplast; M, microspore; ML, middle layer; Nu, nuclei; T, tapetum; Ts, tapetosome; Ve, vacuole. Bars = 5 μm.
Fig 6.
Experimental validation of the quality of the RNA-Seq data by real-time RT-PCR.
The columns indicate the relative RNA levels of selected differentially expressed genes (DEGs) identified between the Ogura-CMS inflorescences and its maintainer inflorescences. The lines show the FPKM expression data of RNA-Seq.
Fig 7.
Expression profiles of differentially expressed genes (DEGs) in the Ogura-CMS and its maintainer fertile (MF) inflorescences of turnip.
(A) Volcano plot showing significantly DEGs with log2 fold change (FC) ≥ 1 or ≤ -1 (Benjamini-Hochberg false discovery rate < 0.05). (B) A hierarchical clustering graph based on the expression values of all significantly DEGs identified in (A).
Table 1.
Functional categories of representative genes significantly up-regulated in inflorescences of the Ogura-CMS line relative to its maintainer fertile (MF) line.
Table 2.
Functional categories of representative genes significantly down-regulated in inflorescences of the Ogura-CMS line relative to its maintainer fertile (MF) line.
Fig 8.
Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.
(A) GO annotations of all unigenes and differential expressed genes (DEGs) in the Ogura-CMS and its maintainer fertile (MF) inflorescences of turnip. The results are summarized in three main categories: biological process, cellular component and molecular function. The y-axis on the right indicates the number of genes in a category. The x-axis on the left indicates the percentage of a specific category of genes in that main category. (B) KEGG pathway annotations of DEGs. (C) KEGG pathway enrichment analysis of DEGs with top 20 enrichment scores. (D) KEGG pathway annotations of the fatty acid degradation pathway. Red marked nodes are associated with up-regulated genes; green nodes are associated with down-regulated genes; blue nodes are associated with both up-regulated and down-regulated genes.
Table 3.
List of known anther and microspore development-involved genes in Arabidopsis and turnip.
Table 4.
List of known anther and microspore development-related genes in turnip and species other than Arabidopsis.
Table 5.
Novel genotype-specific expressed genes with proposed function in male organ development based on GO analyses.