Abstract
Key message This research based on RNA-seq, biochemical, and cytological analyses sheds that ROS may serve as important signaling molecules of cytoplasmic male sterility in CMS-D8 cotton.
Abstract
To understand the mechanism of cytoplasmic male sterility in cotton (Gossypium hirsutum), transcriptomic, cytological, and biochemical analysis were performed between the cytoplasmic male sterility CMS-D8 line, Zhong41A, and its maintainer line Zhong41B. A total of 2335 differentially expressed genes (DEGs) were identified in the CMS line at three different stages of anther development. Bioinformatics analysis of these DEGs indicated their relationship to reactive oxygen species (ROS) homeostasis, including reduction–oxidation reactions and the metabolism of glutathione and ascorbate. At the same time, DEGs associated with tapetum development, especially the transition to secretory tapetum, were down-regulated in the CMS line. Biochemical analysis indicated that the ability of the CMS line to eliminate ROS was decreased, which led to the rapid release of H2O2. Cytological analysis revealed that the most crucial defect in the CMS line was the abnormal tapetum. All these results are consistent with the RNA sequencing data. On the basis of our findings, we propose that ROS act as signal molecules, which are released from mitochondria and transferred to the nucleus, triggering the formation of abnormal tapetum.
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Acknowledgements
We thank Jianbo Cao, Yuan Tan and Fangmei Zhang (Huazhong Agricultural University, China) for their assistance in scanning transmission electron microscopy. This work was supported by grants from National Key Research and Development Program of China (2016YFD0101400), National Natural Science Foundation of China (31371673).
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LY, JW and CX designed the study. JZ and Stewart participated in the experiment material preparation. LY, YW and MZ contributed to experiment conduction, bioinformatics analysis. LY prepared manuscript. JW, CX and SJ participated in the revising the manuscript.
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11103_2018_757_MOESM1_ESM.eps
Supplementary Fig S1. The detailed derivation process of CMS line Zhong41A. Zhong41A was isolated by consecutive repeated backcrosses of tetraploid CMS-D8 line × tetraploid Gossypium hirsutum Zhong41 (Zhong41B) for more than 10 generations. At last, the nucleus of Zhong41A was nearly the same with that of Zhong41B. The only difference of them was existed in the cytoplasm (EPS 738 KB)
11103_2018_757_MOESM2_ESM.eps
Supplementary Fig S2. The cytological section of entire anther development phase of CMS line Zhong41A. Denote: L1, L2 and L3, the three cell layers of the stamen primordia; Ar, archesporial cell; P, parietal layer; Sp, sporogenous layer; E, epidermis; En, endothecium; ML, middle layer; T, tapetum; MMC, microspore mother cell; SMs, shrivelled microsporocytes; DMs, degenerated microsporocytes; NLs, narrowing locules; V, vascular region; StR, stomium region; DLs, degenerated locules. Bars, 50um in (A) to (S) (EPS 9852 KB)
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Supplementary Fig S3. The partial assessment of sequencing data quality. (A) Error rate distribution along reads of six different libraries. The abscissa is the base position of reads. The ordinate is the single-base error rate. (B) Bases content along reads of six different libraries. The abscissa is the base position of reads. The ordinate is the proportion of single-base. The different colors stand for the different base types. (C) Classification of raw reads in six different libraries. The raw reads were classified into clean reads, reads containing N, low quality reads and adaptor reads. Denote: A, Zhong41A; B, Zhong41B, Stage 5, the microspore mother cell stage; Stage 6, the meiosis stage; Stage 7, the tetrad stage (EPS 4528 KB)
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Supplementary Fig S4. The correlation analysis between biological repetitions. R2 stands for the correlation index, which is closer to 1, representing the higher of correlation degree. Denote: A, Zhong41A; B, Zhong41B, 1, the first repetition; 2, the second repetition, Stage 5, the microspore mother cell stage; Stage 6, the meiosis stage; Stage 7, the tetrad stage (EPS 3875 KB)
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Supplementary Fig S5. Changes of transcript abundance levels at stage 5 (A), stage 6 (B) and stage 7(C) between Zhong41A and Zhong41B. Red and green dot refer to Not DEGs and DEGs (EPS 1739 KB)
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Supplementary Fig S6. Heatmap of tapetum development-related genes and other anther development genes. Denote: A, Zhong41A; B, Zhong41B, Stage 5, the microspore mother cell stage; Stage 6, the meiosis stage; Stage 7, the tetrad stage (EPS 2111 KB)
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Supplementary Fig S7. The correlation analysis of gene expression profiles between qRT-PCR and RNA-seq results. The comparison of gene expression levels of 20 selected genes in the Zhong41A plants between qRT-PCR and RNA-seq in analysed stages. Denote: (A) Three stages (B) Stage 5, the microspore mother cell stage (C) Stage 6, the meiosis stage (D) stage 7, the tetrad stage (EPS 2356 KB)
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Yang, L., Wu, Y., Zhang, M. et al. Transcriptome, cytological and biochemical analysis of cytoplasmic male sterility and maintainer line in CMS-D8 cotton. Plant Mol Biol 97, 537–551 (2018). https://doi.org/10.1007/s11103-018-0757-2
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DOI: https://doi.org/10.1007/s11103-018-0757-2