Abstract Ogura-type cytoplasmic male sterility (Ogura-CMS) has been widely used in the hybrid breeding industry for cruciferous vegetables. Turnip (Brassica rapa ssp. rapifera) is one of the most important local cruciferous vegetables in China, cultivated for its fleshy root as a flat disc. Here, morphological characteristics of an Ogura-CMS line ‘BY10-2A’ and its maintainer fertile (MF) line ‘BY10-2B’ of turnip were investigated. Ogura-CMS turnip showed a reduction in the size of the fleshy root, and had distinct defects in microspore development and tapetum degeneration during the transition from microspore mother cells to tetrads. Defective microspore production and premature tapetum degeneration during microgametogenesis resulted in short filaments and withered white anthers, leading to complete male sterility of the Ogura-CMS line. Additionally, the mechanism regulating Ogura-CMS in turnip was investigated using inflorescence transcriptome analyses of the Ogura-CMS and MF lines. The de novo assembly resulted in a total of 84,132 unigenes. Among them, 5,117 differentially expressed genes (DEGs) were identified, including 1,339 up- and 3,778 down-regulated genes in the Ogura-CMS line compared to the MF line. A number of functionally known members involved in anther development and microspore formation were addressed in our DEG pool, particularly genes regulating tapetum programmed cell death (PCD), and associated with pollen wall formation. Additionally, 185 novel genes were proposed to function in male organ development based on GO analyses, of which 26 DEGs were genotype-specifically expressed. Our research provides a comprehensive foundation for understanding anther development and the CMS mechanism in turnip. Introduction As an important and valuable resource, male-sterile varieties are extensively exploited in crop hybrid breeding. Cytoplasmic male-sterility (CMS) is a category of male-sterility resulted from a genomic conflict between the mitochondrial and nuclear genomes, and has been extensively utilized [[40]1]. Various types of CMS have been developed and adopted in plant breeding [[41]2]. It has been proposed that normal microsporogenesis needs appropriate timing of tapetum degeneration and specific gene expression [[42]3]. In CMS system, this elaborate process is complex because of the mitochondrial retrograde signaling pathway and the interaction of nuclear and organelle genomes [[43]4–[44]7]. Considerable variations in morphological phenotype of anther development, particularly of microspore and tapetum behaviors, arise with different nuclear backgrounds and/or cytoplasmic genotype [[45]8]. Mostly, for example, CMS causes the premature degradation of the tapetal cells [[46]9]. However, CMS sorghum and CMS-T maize show a persistent tapetum which likely inhibits nutrient delivery, resulting in the failure of microspore development [[47]8,[48]10]. Ogura-type CMS was first discovered in Japanese radish (Raphanus sativus) and is now widely applied in the breeding of Brassicaceae crops, such as Brassica napus, B. juncea, and B. oleracea, providing a classic model to probe the role of nuclear-cytoplasmic genome interactions [[49]11]. Although interactions of specific mitochondrial gene orf138 with different nuclear backgrounds have been reported to be responsible for Ogura-CMS, diverse floral behaviors attributed to the same cytoplasm in different species have not been fully investigated [[50]11]. For example, anther morphology in Ogura-CMS B. napus is normal, whereas pollen development is impaired and sensitive to temperature [[51]12–[52]16]. However, Ogura-CMS Chinese cabbage (B. rapa ssp. pekinensis) shows reduced plant height and delayed flowering, has shorter filaments, and produces few and infertile pollen grains in indehiscent anthers [[53]17]. All these differences suggest the presence of various regulatory mechanisms and/or multiple regulatory pathways in Brassica spp. Recently, numerous candidate genes involved in CMS have been discovered in different species, such as onion (Allium cepa), cabbage (B. olerace var. capitata), rice (Oryza sativa), and pepper (Capsicum annuum) [[54]7,[55]9,[56]18,[57]19]. In addition, participation of miRNAs and non-coding RNAs is becoming increasingly evident in retrograde regulation of CMS [[58]20–[59]23]. Despite previous extensive work, no specific retrograde pathway has been reported for CMS to date and the regulatory mechanism of CMS is still largely unknown. Exploring the molecular mechanisms underlying CMS is of great importance for improving seed yield in many crop species, especially in crucifers. As a Brassica root crop, turnip (Brassica rapa ssp. rapifera) has been important for human consumption for thousands of years [[60]24]. In this study, the morphological characteristics of an Ogura-CMS line ‘BY10-2A’ and its maintainer fertile (MF) line ‘BY10-2B’ of turnip were investigated, and a detailed RNA sequencing (RNA-Seq) analysis for inflorescences in turnip was conducted. These data provide a comprehensive view on the dynamic gene expression networks and their potential roles in controlling anther development. Using pairwise comparisons, we identified 5,117 DEGs, which might respond to the mutation of the mitochondrial ORF138 locus. Among them, 185 novel genes were proposed to function in male organ development based on GO analyses. These findings provide a comprehensive insight into the regulatory networks responsible for Ogura-CMS tapetum abnormality and pollen abortion in turnip, and demonstrate that cytoplasmic retrograde regulation is probably a principal molecular mechanism for CMS in turnip. Materials and methods Plant materials and growth conditions Previously, the Ogura-CMS line ‘BY10-2A’ of B. rapa ssp. rapifera was developed by inter-specific hybridization between B. rapa ssp. chinensis as the Ogura-CMS cytoplasm donor and fertile B. rapa L. ssp. rapifera, followed by 10 recurrent generations of back-crossing. The Ogura-CMS line and its maintainer fertile (MF) line ‘BY10-2B’ were cultivated in the experimental farm of Wenzhou Vocational College of Science and Technology, Wenzhou, Zhejiang, China. Plant morphological analysis and floret structure observation Plants were observed and photographed at 32, 48, 110, and 180 days after germination. The length and diameter of fleshy roots were measured at 110 and 180 days after germination. A week after the first anthesis, florets of both the Ogura-CMS and MF lines were collected. The floret structures were observed under a Leica MZ16FA stereoscopic microscope (Leica Microsystems, Wetzlar, Germany). Floral buds, floral organs, anthers and pollen grains morphological analysis Ogura-CMS and MF floral buds at different anther developmental stages, and various floral organs were fixed with 2.5% glutaraldehyde in phosphate buffer (pH 7.0) overnight, and post-fixed with 1% OsO[4] in phosphate buffer for 1 h. Subsequently, the specimens were dehydrated in a graded ethanol series (50%, 70%, 80%, 90%, 95%, 2×100%). For scanning electron microscopy, the dehydrated specimens were coated with gold-palladium in an Eiko Model IB5 ion coater (Eiko Engineering Company, Ibaraki, Japan), and photographed in a Hitachi Model TM-1000 scanning electron microscope (Tokyo, Japan) [[61]http://dx.doi.org/10.17504/protocols.io.zz4f78w]. For semi-thin section analyses, the dehydrated specimens were embedded in Spurr resin. Semi-thin sections (1 μm) were sliced under a LKB 11800 PYRAMITOME ultramicrotome (Stockholm, Sweden) and stained with 0.5% toluidine blue. Images of anther cross-sections were obtained with a Leica DMLB fluorescence microscope (Leica Microsystems, Wetzlar, Germany) [[62]http://dx.doi.org/10.17504/protocols.io.zz5f786]. For transmission electron microscopy (TEM), ultrathin sections (70 nm) were obtained and stained with uranyl acetate followed by alkaline lead citrate, and photographed with a Hitachi Model H-7650 transmission electron microscope (Tokyo, Japan) [[63]http://dx.doi.org/10.17504/protocols.io.zz6f79e]. RNA sample collection and total RNA isolation After flowering, all floral buds of an inflorescence from the Ogura-CMS and MF lines of B. rapa ssp. rapifera were collected. In each case, samples were harvested and pooled from ten individual plants with transcriptome profiles representing ‘f’ difference, then immediately frozen in liquid nitrogen and stored at -70°C until RNA isolation. For biological repetitions, RNA was extracted from three samples using the EASYspin Plant RNA kit (Aidlab Biotechnologies Corporation, Bejing, China). RNA quality and quantity were characterized on a 1% agarose gel, and determined with a NanoPhotometer spectrophotometer (IMPLEN, CA, USA) and a Qubit RNA Assay Kit in Qubit2.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Illumina sequencing and de novo transcriptome assembly A total of 3 μg RNA per sample was used for library preparation using a NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA). The library preparations were then sequenced on an Illumina Hiseq 2000 platform by the Biomarker Biotechnology Corporation (Beijing, China). Clean reads were filtered from the raw reads by removing reads containing adapter, poly-N and low-quality reads. All the downstream analyses were based on clean data with quality determined by Q20, Q30, GC-content and sequence duplication levels. Then de novo transcriptome assembly was accomplished using the Trinity platform [[64]25] with min-kmer-cov set to 2 by default and all other parameters set to default. The RNA-Seq data were uploaded to the Sequence Read Archive of the National Center for Biotechnology Information (NCBI) (DOI: [65]https://www.ncbi.nlm.nih.gov/sra/PRJNA505114; accession number: PRJNA505114). Annotation of differentially expressed genes Differentially expressed genes (DEGs) were screened out using the DESeq (2010) R Package. Genes with a Benjamini-Hochberg false discovery rate (FDR) < 0.05 and a log[2] fold change (FC) ≥ 1 or ≤ -1 in each pairwise comparison were assigned as differential expressed. DEG sequences were blast in the NCBI non-redundant protein (Nr) database, Swiss-Prot database and orthologous groups of genes (eggNOG) database, and also aligned to the Clusters of Orthologous Group (COG), Clusters of Protein homology database (KOG), and Homologous protein family (Pfam) database to predict and classify functions [[66]26,[67]27]. Gene ontology (GO) enrichment analysis of DEGs was performed by the topGO (2007) R packages based on a Kolmogorov-Smirnov test. Pathway analysis of DEGs was carried out to detect the important pathways, based on the database of Kyoto Encyclopedia of Genes and Genomes (KEGG). Statistical enrichment of DEGs in KEGG pathways was identified by KOBAS software using a hypergeometric test [[68]28]. Real-time reverse transcription polymerase chain reaction (RT-PCR) validation Sixteen DEGs were randomly selected for validation using real-time RT-PCR. Residual RNA samples for DEG analysis were transcribed into cDNA with a HiScrip II 1^st Strand cDNA Synthesis Kit (Vazyme Biotech, Nanjing, China) as the template. ACT7 was used as the normalization control [[69]29]. Real-time RT-PCR was performed on an Applied Biosystems 7500 Real-time PCR System (ThermoFisher, MA, USA) and the relative expression levels were analyzed. Three technical repeats were performed. All primers used were listed in [70]S1 Table. Results The Ogura-CMS turnip displays complete male sterility Despite 10 generations of back-crossings, Ogura-CMS plants showed a reduction in fleshy root size compared with its MF line ([71]Fig 1A–1C). Furthermore, during the reproductive growth phase, the Ogura-CMS line was distinguishable from the MF line by its short filaments and withered white anthers ([72]Fig 1D and 1E). The floral buds from the Ogura-CMS line showed the same developmental pattern as those of its MF line ([73]Fig 2A and 2C). However, compared with the yellow and plump anthers of the MF line ([74]Fig 2B), no pollen was observed in the mature Ogura-CMS anthers ([75]Fig 2D). Other than pollen absence and stamen abnormality, floral organs presented normal morphologies ([76]S1 Fig), including female gametophytes as pollinating the MF pollen grains on the Ogura-CMS stigma led to normal silique growth and full seed set ([77]S2 Fig). Fig 1. Morphological features of fleshy roots and flowers in the Ogura-CMS line and its maintainer fertile (MF) line of turnip. [78]Fig 1 [79]Open in a new tab (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. [80]Fig 2 [81]Open in a new tab (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). Aberrant anther development occurs during transition from microspore mother cells to tetrads To determine the precise stage at which Ogura-CMS anther shrinkage begins, semi-thin sections of anthers from various developmental stages were prepared and further analyzed by microscopy. No differences in microspore development were observed inside the anther locules during the process of meiotic division up to the tetrad stage ([82]Fig 3A, 3B, [83]3F and 3G). Each Ogura-CMS tetrad ([84]Fig 3G) contained four microspores, similar to those of the MF line ([85]Fig 3B), indicating that meiosis is normal in the Ogura-CMS line. Both Ogura-CMS and MF tetrads were surrounded by a tapetum, a middle layer, an endothecium, and an epidermis from the inside out at the tetrad stage ([86]Fig 3B and 3G). However, it was noticeable that the Ogura-CMS tapetum ([87]Fig 3G) swelled at the center of the locule, and the cytoplasm was distinguishably clear from that of the MF line ([88]Fig 3B). After the tetrad stage, the MF middle layer degenerated, then disappeared by the uninucleate stage, and the MF anthers released microspores that developed into mature pollen grains ([89]Fig 3C–3E). However, the collapse of the Ogura-CMS microspores started at the uninucleate microspore stage and was accompanied by extensive degeneration ([90]Fig 3H). Moreover, the middle layer persisted at this point, together with significantly enlarged tapetum, crushing the free microspores to the locule center. At later stages of development, the collapse of the microspore was even more remarkable due to the degeneration of its entire contents ([91]Fig 3I). Eventually, defective microspore development and early clearing of tapetal cytoplasm led to shrunken anthers with collapsed locules and remnants of pollen grains adhered to the inner face of the epidermis ([92]Fig 3J). Fig 3. Anther and microspore development in the Ogura-CMS line and its maintainer fertile (MF) line of turnip. [93]Fig 3 [94]Open in a new tab (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. Abnormal Ogura-CMS microspore development was further confirmed by TEM. Ogura-CMS microspores underwent similar development to those of MF from the microspore mother cell stage to the tetrad stage ([95]Fig 4A, 4B, 4F and 4G) and, at the uninucleate stage, were distinguishable from MF microspores ([96]Fig 4C and 4H). At the uninucleate stage, the MF microspores had almost complete basic intine and exine structure ([97]Fig 4C). Exine was comprised of inner nexine and outer sexine. Sexine further possessed a three-dimensional structure composed of baculae and a roof-like tectum, whereas the bilayer nexine, consisting of nexine I and nexine II, was laid down on the intine layer ([98]Fig 4K). Subsequently, bicellular microspores were generated concurrently with the size increase of exine, and the mature exine structure was visually completed at this point ([99]Fig 4D and 4L). Finally, the mature pollen grain was complete with tryphine ([100]Fig 4E and 4M). However, after dissolution of the callose wall, free Ogura-CMS microspores were deformed with a thin and incomplete exine layer ([101]Fig 4H). The nexine I was the last layer overlying the microspore plasma membrane but not the intine layer ([102]Fig 4N). By the bicellular stage, with an empty body, pollen grains were prepared with incomplete-developed layers of exine and tryphine ([103]Fig 4O), which developed into remnants at the dehiscent stage ([104]Fig 4J). Fig 4. Transmission electron microscopy observation of microspore development in the Ogura-CMS line and its maintainer fertile (MF) line of turnip. [105]Fig 4 [106]Open in a new tab (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). Coordinated with microspore development, visible changes occurred to the surrounding walls in the anther locules and were also observed when using TEM. No defects on Ogura-CMS surrounding walls were detected at the microspore mother cell stage ([107]Fig 5F) compared with those of the MF line ([108]Fig 5A). Anther primordia which were enclosed in an epidermis, differentiated inwardly into the endothecium, middle layer, tapetum, and microspore mother cells. At the tetrad stage, MF tapetal cells became mature and vacuolated, with a heterogeneous density in the cytoplasm ([109]Fig 5B). However, the anther pattern was significantly altered in Ogura-CMS tapetum at this stage. Ogura-CMS tapetal cells enlarged and swelled to expand to the center of the locules, with larger vacuoles and a clearing cytoplasm ([110]Fig 5G). When the callose wall totally dissolved and free individual microspores released into the anther locules, a large number of elaioplasts emerged in the MF tapetum, and remnants of the middle layer were absent in the MF anther ([111]Fig 5C), but still clearly present in the Ogura-CMS anther ([112]Fig 5H). In addition, Ogura-CMS tapetal cells were full of shedding materials such as tapetosomes and significantly swelled, crushing the free microspores to the locule center ([113]Fig 5H). This premature degradation of the tapetum was obvious, which fulfilled the programmed cell death ahead of schedule. All that remained of the Ogura-CMS tapetum was an almost empty shell at the bicellular stage ([114]Fig 5I), but integral tapetal cells with a large amount of elaioplasts were still observed in the MF anther ([115]Fig 5D). Moreover, the endothecium also appeared abnormal in the Ogura-CMS anther, devoid of any content in the cytoplasm at the bicellular stage ([116]Fig 5I), and had disappeared completely at the mature pollen stage ([117]Fig 5J). Fig 5. Transmission electron microscopy observation of tapetum development in the Ogura-CMS line and its maintainer fertile (MF) line of turnip. [118]Fig 5 [119]Open in a new tab (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. Overall, the Ogura-CMS anthers showed two distinct defects that occurred during the transition from microspore mother cells to tetrads: the failure of microspore development and the swollen tapetum layer. Defective microspore production and premature tapetum degeneration during microgametogenesis led to complete male sterility of the Ogura-CMS line. RNA-Seq analysis on inflorescences of the Ogura-CMS line and its maintainer fertile line in turnip To explore the molecular basis for the morphological differences in anther and microspore development described above, RNA-Seq analyses were conducted to generate transcriptome profiles of the whole inflorescences from the Ogura-CMS and MF lines. RNA-Seq analysis was performed with three biological replicates for each. After removing low-quality reads, an average of 24.2 × 10^6 clean reads per library were generated ([120]S2 Table). The de novo assembly resulted in a total of 84,132 unigenes ([121]S3 Fig and [122]S3 Table), which were proposed to be expressed during floral bud development in turnip. To verify the quality of the RNA-Seq data, real-time RT-PCR analysis was conducted on 16 randomly selected genes ([123]Fig 6). The strong correlation between the RNA-Seq and real-time RT-PCR results indicated high reliability of our transcriptomic profiling data. Fig 6. Experimental validation of the quality of the RNA-Seq data by real-time RT-PCR. [124]Fig 6 [125]Open in a new tab 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. To determine whether the genes regulating pollen and tapetum development are defective or expressed abnormally, differential expression of genes during thereproductive development of the MF and CMS lines was analyzed. The reads were mapped to unigenes and quantified to show gene expression abundance by Fragments Per Kilobase of transcript per Million mapped reads (FPKM) [[126]30]. The FPKM expression data were tested by correlation analysis to evaluate sampling between biological replicates, and all correlation coefficients were ≥ 0.82. Using the DESeq (2010) R Package with a FDR < 0.05 and a log[2] FC ≥ 1 or ≤ -1, pairwise comparisons of Ogura-CMS versus MF showed that 5,117 genes were significantly differentially expressed, of which 1,339 genes were significantly up-regulated and 3,778 genes significantly down-regulated in the Ogura-CMS line relative to the MF line ([127]Fig 7). Representative genes for the up- and down-regulated DEGs are listed in Tables [128]1 and [129]2, respectively, according to their functional categories. Referencing to the chromosome of Chinese cabbage (Brassica rapa ssp. pekinensis), the DEGs are widely distributed in all chromosomes ([130]S4 Fig). Fig 7. Expression profiles of differentially expressed genes (DEGs) in the Ogura-CMS and its maintainer fertile (MF) inflorescences of turnip. [131]Fig 7 [132]Open in a new tab (A) Volcano plot showing significantly DEGs with log[2] 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. Gene ID log[2] fold change (Ogura-CMS line/MF line) Description Transcription factors TRINITY_DN24247_c4_g1 4.989846 Transcription factor MYB39 TRINITY_DN22677_c1_g9 3.653598 Probable WRKY transcription factor 71 TRINITY_DN25088_c3_g1 3.295980 NAC transcription factor 29 TRINITY_DN22677_c1_g2 3.196358 Probable WRKY transcription factor 71 TRINITY_DN23536_c1_g7 3.049643 Homeobox-leucine zipper protein ATHB-21 Carbohydrate transport and metabolism TRINITY_DN24721_c0_g1 5.481076 β-glucosidase 27 TRINITY_DN22492_c0_g5 5.408367 Peroxidase 15 TRINITY_DN24408_c2_g2 4.629681 Probable xyloglucan endotransglucosylase/hydrolase protein 18 TRINITY_DN22492_c0_g7 4.542994 Peroxidase TRINITY_DN22492_c0_g3 3.409620 Peroxidase 49 Lipid transport and metabolism TRINITY_DN25317_c1_g5 1.323946 Fatty acyl-CoA reductase 1 TRINITY_DN24410_c0_g2 1.435586 Probable acyl-activating enzyme 16 TRINITY_DN25317_c1_g16 1.800464 Fatty acyl-CoA reductase 1 Plant hormone signal transduction TRINITY_DN22137_c0_g2 2.385851 Auxin-induced protein X15 TRINITY_DN24146_c0_g1 2.268715 Indole-3-acetic acid-amido synthetase GH3.5 TRINITY_DN24009_c3_g3 1.789990 Indole-3-acetic acid-induced protein ARG7 TRINITY_DN26266_c0_g2 1.751535 Serine/threonine-protein kinase SRK2J TRINITY_DN23900_c1_g2 1.737397 Auxin-responsive protein IAA12 [133]Open in a new tab Table 2. Functional categories of representative genes significantly down-regulated in inflorescences of the Ogura-CMS line relative to its maintainer fertile (MF) line. Gene ID log[2] fold change (Ogura-CMS line/MF line) Description Transcription factors TRINITY_DN25469_c0_g5 -8.944461 Zinc finger protein ZAT2 TRINITY_DN24004_c2_g7 -8.817322 NAC transcription factor 25 TRINITY_DN23291_c0_g9 -6.858249 Transcription factor GAMYB TRINITY_DN25812_c2_g1 -6.813564 MADS-box transcription factor 16 TRINITY_DN22189_c1_g8 -6.333698 Probable WRKY transcription factor 31 Carbohydrate transport and metabolism TRINITY_DN27564_c2_g4 -12.994995 Exopolygalacturonase clone GBGA483 TRINITY_DN22409_c1_g6 -12.479006 Probable pectate lyase 4 TRINITY_DN26448_c4_g4 -11.859968 Pectinesterase 21 TRINITY_DN23204_c0_g1 -11.382811 Pectinesterase PPME1 TRINITY_DN27059_c3_g2 -11.281507 Xyloglucan endotransglucosylase/hydrolase protein 3 Lipid transport and metabolism TRINITY_DN24821_c1_g3 -9.749978 Delta(8)-fatty-acid desaturase 2 TRINITY_DN26166_c1_g2 -8.847981 Dehydrodolichyl diphosphate synthase 8 TRINITY_DN18964_c0_g2 -8.041336 Probable lipid phosphate phosphatase 4 TRINITY_DN22477_c0_g1 -7.414755 Long chain acyl-CoA synthetase 5 TRINITY_DN26830_c1_g1 -7.196643 Phosphoinositide phospholipase C 6 Plant hormone signal transduction TRINITY_DN22372_c0_g1 -9.961644 Indole-3-acetic acid-amido synthetase GH3.17 TRINITY_DN17602_c0_g1 -6.559842 4-substituted benzoates-glutamate ligase GH3.12 TRINITY_DN22992_c3_g4 -5.265387 Auxin-induced protein 15A TRINITY_DN27694_c2_g5 -2.799438 ABSCISIC ACID-INSENSITIVE 5-like protein 1 [134]Open in a new tab Functional annotation of differentially expressed genes during reproductive development of the Ogura-CMS line and its maintainer fertile line of turnip To perform annotation analysis of the DEGs, eight public databases including COG, GO, KEGG, Swiss-prot database, KOG, Pfam, eggNOG, and Nr were searched. In total, 4,864 DEGs were found and annotated in detail in at least one of these databases ([135]S4 Table). To forecast functional classifications of annotated DEGs, the GO and KEGG pathway analyses were performed to provide a clue. We utilized all DEGs for GO analysis and found that 76% of DEGs (3,889 out of 5,117) have at least one GO term assigned and were categorized into 46 functional groups ([136]Fig 8A). Among these, the top three dominant categories were involved in cellular, metabolic, and single-organism processes ([137]Fig 8A). The KEGG pathway analysis manifested that 50 pathways were significantly enriched, particularly metabolic pathways, plant-pathogen interaction, and plant hormone signal transduction ([138]Fig 8B). The DEGs were found to be mostly enriched in ether lipid metabolism ([139]Fig 8C). Furthermore, many genes involved in fatty acid metabolism which is essential for the assembly of exine and tryphine [[140]3,[141]31] were dysregulated ([142]Fig 8D). Fig 8. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. [143]Fig 8 [144]Open in a new tab (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. In all annotated DEGs, 1,289 genes were significantly up-regulated and 3,575 genes were significantly down-regulated in the Ogura-CMS relative to MF inflorescences ([145]S4 Table). Among these, 610 DEGs were specifically expressed in the MF inflorescences and only 31 DEGs in the Ogura-CMS inflorescences ([146]S5 and [147]S6 Tables), implying that there is a considerable scope for further research to discover novel CMS-associated genes in turnip. In addition, several genes that were classified as function unknown but specifically expressed in the MF inflorescences, such as TRINITY_DN22922_c0_g2, TRINITY_DN37525_c0_g1, and TRINITY_DN23102_c0_g1, could be good candidates for CMS-related genes. Genes related to anther development and microspore formation Based on genetic and transcriptomic studies, it has been long assumed that Arabidopsis pollen development involves precise spatial and temporal cooperation of the tapetum and the gametophyte itself, and relies on the functions of numerous genes and their dynamic regulatory network [[148]32]. We compared the expressive alteration of homologs of Arabidopsis genes previously reported to be associated with anther and pollen development, to unravel whether cytoplasmic retro-regulated counterparts of those genes from the nucleus. In addition, some functionally known genes involved in this unique process in species other than Arabidopsis were also compared. As the innermost layer surrounding the sporogenous cells in the anther, the tapetum provides not only energy, but also nutrients, metabolites, and sporopollenin precursors for microspore development [[149]33]. Coordinated with the defective tapetum, homologs of some extensively demonstrated genes and enzymes associated with tapetum development in Arabidopsis ([150]Table 3) and other species ([151]Table 4) exhibited altered expression in Ogura-CMS. For example, AMS is a basic helix-loop-helix (bHLH) transcription factor, one of the master regulators for tapetum and microspore development in Arabidopsis [[152]3]. Expression of a counterpart of AMS (TRINITY_DN27860_c1_g1) was down-regulated in Ogura-CMS. Some turnip homologs of AMS-dependent genes including QRT3 (TRINITY_DN22468_c2_g3), CYP98A8 (TRINITY_DN26854_c1_g2), CHS (TRINITY_DN27063_c0_g1), EXL6 (TRINITY_DN24807_c1_g1, and TRINITY_DN26730_c0_g3), and PAB5 (TRINITY_DN22996_c2_g9, TRINITY_DN22996_c2_g5, and TRINITY_DN22996_c2_g10), showed reduced expression, but some genes did not, such as CYP703A2, CYP704B1, KCS7, LAP5 and LTP12 ([153]Table 3). It was noteworthy that counterparts of two AMS-dependent genes, TKPR1 (TRINITY_DN23571_c0_g2, TRINITY_DN25196_c0_g1, and TRINITY_DN23571_c0_g1) and A6 (TRINITY_DN25127_c0_g3), suggested to be directly regulated by AMS in Arabidopsis, had increased expression in Ogura-CMS ([154]Table 3). In addition, AMS regulatory pathway oriented analyses showed that turnip homologs of ATA20 (TRINITY_DN25943_c1_g3 and TRINITY_DN25943_c1_g1), bHLH89 (TRINITY_DN24940_c2_g5), and bHLH91 (TRINITY_DN22984_c1_g2 and TRINITY_DN22984_c1_g1) were down-regulated ([155]Table 3), but DYT1, TDF1, MS188, and MS1 were not in the pool of DEGs. Table 3. List of known anther and microspore development-involved genes in Arabidopsis and turnip. Gene ID log[2] fold change (Ogura-CMS line/ maintainer line) Up/down-regulation (Ogura-CMS line/ maintainer line) Arabidopsis References