Comparative Transcriptome Analysis Reveals a Potential Regulatory Network for Ogura Cytoplasmic Male Sterility in Cabbage (Brassica oleracea L.)

Abstract

Ogura cytoplasmic male sterility (CMS) lines are widely used breeding materials in cruciferous crops and play important roles in heterosis utilization; however, the sterility mechanism remains unclear. To investigate the microspore development process and gene expression changes after the introduction of orf138 and Rfo, cytological observation and transcriptome analysis were performed using a maintainer line, an Ogura CMS line, and a restorer line. Semithin sections of microspores at different developmental stages showed that the degradation of tapetal cells began at the tetrad stage in the Ogura CMS line, while it occurred at the bicellular microspore stage to the tricellular microspore stage in the maintainer and restorer lines. Therefore, early degradation of tapetal cells may be the cause of pollen abortion. Transcriptome analysis results showed that a total of 1287 DEGs had consistent expression trends in the maintainer line and restorer line, but were significantly up- or down-regulated in the Ogura CMS line, indicating that they may be closely related to pollen abortion. Functional annotation showed that the 1287 core DEGs included a large number of genes related to pollen development, oxidative phosphorylation, carbohydrate, lipid, and protein metabolism. In addition, further verification elucidated that down-regulated expression of genes related to energy metabolism led to decreased ATP content and excessive ROS accumulation in the anthers of Ogura CMS. Based on these results, we propose a transcriptome-mediated induction and regulatory network for cabbage Ogura CMS. Our research provides new insights into the mechanism of pollen abortion and fertility restoration in Ogura CMS.

Keywords: Ogura CMS; cabbage; comparative transcriptome analysis; pollen development.

Figure 1

Flower morphology and pollen viability of the maintainer line, Ogura CMS line, and restorer line. (A,D) Maintainer line 19–616. (B,E) Ogura CMS 19–2167. (C,F) Restorer line FR2202. Bar = 0.5 cm.

Figure 2

Cytological observation of anthers in the maintainer line, Ogura CMS line, and restorer line. (A–F) Maintainer line 19–616. (G–L) Ogura CMS 19–2167. (M–R) Restorer line FR2202. (A,G,M) Microsporocyte stage. (B,H,N) Meiotic stage. (C,I,O) Tetrad stage. (D,J,P) Uninucleate stage. (E,K,Q) Bicellular microspore stage to trinucleate microspore stage. (F,L,R) Mature pollen stage. Bar = 50 µm. PMC, pollen mother cell; Ep, epidermis; En, endothecium; ML, middle layer; T, tapetum; Td, tetrad; Ms, microspore; MP, mature pollen.

Figure 3

Differential expression analysis of nuclear genes. (A–C) The pairwise comparison between 19–616, 19–2167 and FR2202. (D) Analysis of the shared DEGs in 19–616, 19–2167 and FR2202.

Figure 4

Gene ontology analysis of the core DEGs enriched in part IV. (A) The number of DEGs enriched in the three categories. (B) The expression trends and annotations of the core DEGs.

Figure 5

The expression trends and annotations of DEGs enriched in pollen development (A); energy metabolism (B); carbohydrate, lipid, and protein metabolism (C); and transcription factors (D).

Figure 6

Mitochondrial biochemical indices among three lines. (A) ATP content. (B) ATPase activity. (C) H₂O₂ content. Significant differences between samples labeled with different Roman letters (a, b) were determined by Turkey’s tests. p < 0.05.

Figure 7

Potential transcriptome-mediated regulation network in cabbage Ogura CMS. PCD, programmed cell death; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PDC, pyruvate dehydrogenase; CS, citrate synthase; ND, NADH dehydrogenase; SDH, succinate dehydrogenase; CCO, cytochrome c oxidase; ATG8, autophagy-related protein 8; ACD11, accelerated cell death 11; SHT, spermidine hydroxycinnamoyl transferase; CCM, caffeoyl-CoA O-methyltransferase; BGL, Beta-glucosidase; AGP, arabinogalactan protein; STP, sugar transport protein; PME, pectinesterase; PMEI, Pectinesterase inhibitor; NLT, non-specific lipid-transfer protein; PEAM, phosphoethanolamine N-methyltransferase; Ole, Oleosin.