Comparative Transcriptome Analysis Reveals Key Insights into Fertility Conversion in the Thermo-Sensitive Cytoplasmic Male Sterile Wheat

Abstract

Thermo-sensitive cytoplasmic male sterility (TCMS) plays a crucial role in hybrid production and hybrid breeding; however, there are few studies on molecular mechanisms related to anther abortion in the wheat TCMS line. In this study, FA99, a new wheat thermo-sensitive cytoplasmic male sterility line, was investigated. Fertility conversion analysis showed that FA99 was mainly controlled by temperature, and the temperature-sensitive stage was pollen mother cell formation to a uninucleate stage. Further phenotypic identification and paraffin section showed that FA99 was characterized by indehiscent anthers and aborted pollen in a sterile environment and tapetum was degraded prematurely during the tetrad period, which was the critical abortion period of FA99. The contents of O^2-, H₂O₂, MDA and POD were significantly changed in FA99 under a sterile environment by the determination of physiological indexes. Furthermore, through transcriptome analysis, 252 differentially expressed genes were identified, including 218 downregulated and 34 upregulated genes. Based on KOG function classification, GO enrichment and KEGG pathways analysis, it was evident that significant transcriptomic changes in FA99 under different fertility environments, and the major differences were "phenylalanine metabolism", "phenylpropanoid biosynthesis", "cutin, suberine and wax biosynthesis", "phenylalanine, tyrosine and tryptophan biosynthesis" and "citrate cycle (TCA cycle)". Finally, we proposed an intriguing transcriptome-mediated pollen abortion and male sterility network for FA99. These findings provided data on the molecular mechanism of fertility conversion in thermo-sensitive cytoplasmic male sterility wheat.

Keywords: cytoplasmic male sterility; fertility conversion; hybrid wheat; transcriptome sequencing.

Figure 1

Phenotypic characteristics of FA99 and J411 in different fertility conditions. (A,E) B-FA99 (FA99 in the fertile condition); (B,F) D-FA99 (FA99 in the sterile condition); (C,G) B-J411 (J411 in the fertile condition); (D,H) D-J411 (J411 in the sterile condition); (A–D) Floret at the trinucleate stage; (E–H) I₂-KI staining of pollen grains. Scale bars = 500 µm (A–D); 100 µm (E–H).

Figure 2

Comparative analysis of paraffin sections of FA99 and J411 anthers at different stages in different fertility environments. MMCs: microspore mother cell stage; DYAd: dyad stage; Td: tetrad stage; Un: uninucleate stage; Bn: binucleate stage; Tn: trinucleate stage; E: Epidermis; En: Endothecium; ML: Middle layer; T: Tapetum; Msp: Microspores; Scale bars = 50 µm.

Figure 3

Analysis of FA99-related physiological indexes in different fertility environments. (A) O²⁻ contents; (B) H₂O₂ contents; (C) MDA contents; (D) POD activity. MMCs: microspore mother cell stage; DYAd: dyad stage; Td: tetrad stage; Un: uninucleate stage; Bn: binucleate stage; Tn: trinucleate stage. Students’ t test * p < 0.05, ** p < 0.01. Each value represents the mean ± SD (n = 3).

Figure 4

Analysis of DEGs between the samples. (A) Venn diagram of all DEGs; (B) Differences in the abundance of genes.

Figure 5

Gene Ontolgoy (GO) classifications of differentially expressed genes (DEGs) (A) and the Z-score bubble chart of GO enrichment analysis (B) in B-FA99 and D-FA99 during the tetrad stage. The ordinate is -log10 (p value), the abscissa is the up–down normalization value (the ratio of the difference between the number of differentially upregulated genes and the number of differentially downregulated genes to the total differential genes). The bubble size indicates the number of target genes enriched by the current GO term; the yellow line represents the threshold of p value = 0.05; the right side is the list of terms with the top 20 p values, and different colors represent different ontology.

Figure 6

Primary and secondary classification (A) and the top 20 significantly enriched pathways based on KEGG (B,C). C: cellular processes; O: organismal systems; G: genetic information processing; E: environmental information processing; M: metabolism.

Figure 7

Heatmap showing the relative expression level of the nine key genes determined by RNA-seq analysis (A) and qRT-PCR (B).

Figure 8

Possible transcriptome-mediated male sterility network in FA99. UGT, UDP-glucose glucosyltransferase; bglX, β-glucosidase; CS, citrate synthase; MDH, malate dehydrogenase, SDH, succinate dehydrogenase; αKG-DH, 2-oxoglutarate dehydrogenase E1 component; IDH₂, isocitrate dehydrogenase; ACO, aconitate hydratase; FAD, flavin adenine dinucleotide; FADH₂, flavine adenine dinucleotide, reduced; NAD, nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide; TAT, tyrosine aminotransferase; PAL, phenylalanine ammonia-lyase; POD, peroxidase; PXG, peroxygenase; ROS, reactive oxygen species; I, NADH dehydrogenase; II, succinate dehydrogenase; III, cytochrome bc1 complex; IV, cytochrome c oxidase; V, ATP synthase.