Integrated Cytological, Physiological, and Comparative Transcriptome Profiling Analysis of the Male Sterility Mechanism of 'Xinli No.7' Pear (Pyrus sp.)

Abstract

Pyrus bretschneideri 'Xinli No.7', a progeny of Pyrus sinkiangensis 'Korla Fragrant Pear', is an early-maturing, high-quality pear (Pyrus spp.) cultivar. As a dominant variety in China's pear-producing regions, it holds significant agricultural importance. Investigating its male sterility (MS) mechanisms is critical for hybrid breeding and large-scale cultivation. Integrated cytological, physiological, and transcriptomic analyses were conducted to compare dynamic differences between male sterility (MS, 'Xinli No.7') and male-fertile (MF, 'Korla Fragrant Pear') plants during anther development. Cytological observations revealed that, compared with 'Korla Fragrant Pear', the tapetum of 'Xinli No.7' exhibited delayed degradation and abnormal thickening during the uninucleate microspore stage. This pathological alteration compressed the microspores, ultimately leading to their abortion. Physiological assays demonstrated excessive reactive oxygen species (ROS) accumulation, lower proline content, higher malondialdehyde (MDA) levels, and reduced activities of antioxidant enzymes (peroxidase and catalase) in MS plants. Comparative transcriptomics identified 283 co-expressed differentially expressed genes (DEGs). Functional enrichment linked these DEGs to ROS-scavenging pathways: galactose metabolism, ascorbate and aldarate metabolism, arginine and proline metabolism, fatty acid degradation, pyruvate metabolism, and flavonoid biosynthesis. qRT-PCR validated the expression patterns of key DEGs in these pathways. A core transcriptome-mediated MS network was proposed, implicating accelerated ROS generation and dysregulated tapetal programmed cell death. These findings provide theoretical insights into the molecular mechanisms of male sterility in 'Xinli No.7', supporting future genetic and breeding applications.

Keywords: ROS-scavenging systems; male sterility; pear; transcriptome; ‘Xinli No.7’.

Figure 1

A paraffin section observation of the pollen and anther wall development of ‘Korla Fragrant Pear’ (left) and ‘Xinli No.7’ (right). ST, swollen tapetum; T, tapetum; Tds, tetrads; MSp, microspores; DMsp, deformed microspore. The scale is 20 μm. (A). Following meiosis, the pollen mother cells of ‘Korla Fragrant Pear’ form tetrads that are dispersed within the anther locules. (B). During mitotic division of microspores, tapetal cells undergo progressive degeneration to supply essential nutrients for microspore development, while the microspores migrate toward the anther wall. (C). At pollen maturity, the tapetum has completely degenerated, and the remaining middle layer cells are compactly arranged to form a smooth inner lining of the anther locules. (D). The microspores gradually enlarge and become rounded, with thickening of the exine, ultimately forming spherical pollen grains. (E). Following meiosis, the pollen mother cells of ‘Xinli No.7’ form tetrads that are dispersed within the anther locules. ‘Xinli No.7’ tetrad period. (F). The tapetal cells neither separate from the middle layer cells nor undergo degeneration or degradation. Instead, they continue to proliferate and gradually encroach into the locule space. (G). The tapetum and middle-layer cells undergo excessive abnormal development, resulting in the compression and obliteration of the anther locules, while the microspores undergo degradation. (H). ‘Xinli No.7’ pollen vacuolization.

Figure 1

A paraffin section observation of the pollen and anther wall development of ‘Korla Fragrant Pear’ (left) and ‘Xinli No.7’ (right). ST, swollen tapetum; T, tapetum; Tds, tetrads; MSp, microspores; DMsp, deformed microspore. The scale is 20 μm. (A). Following meiosis, the pollen mother cells of ‘Korla Fragrant Pear’ form tetrads that are dispersed within the anther locules. (B). During mitotic division of microspores, tapetal cells undergo progressive degeneration to supply essential nutrients for microspore development, while the microspores migrate toward the anther wall. (C). At pollen maturity, the tapetum has completely degenerated, and the remaining middle layer cells are compactly arranged to form a smooth inner lining of the anther locules. (D). The microspores gradually enlarge and become rounded, with thickening of the exine, ultimately forming spherical pollen grains. (E). Following meiosis, the pollen mother cells of ‘Xinli No.7’ form tetrads that are dispersed within the anther locules. ‘Xinli No.7’ tetrad period. (F). The tapetal cells neither separate from the middle layer cells nor undergo degeneration or degradation. Instead, they continue to proliferate and gradually encroach into the locule space. (G). The tapetum and middle-layer cells undergo excessive abnormal development, resulting in the compression and obliteration of the anther locules, while the microspores undergo degradation. (H). ‘Xinli No.7’ pollen vacuolization.

Figure 2

Changes in physiological indexes of anthers of ‘Xinli No.7’ and ‘Korla Fragrant Pear’ in different periods: (A). Hydrogen peroxide content (B). Malondialdehyde content (C). Proline content (D). Amino acid content (E). SOD activity (F). POD activity (G). CAT activity. Values are means ± SD of three replicates. Asterisks represent statistically significant differences between ‘Xinli No.7’ and ‘Korla Fragrant Pear’ (Student’s t-test * p < 0.05, ** p < 0.01).

Figure 3

(A). Correlation analysis between samples, (B). principal component analysis between samples, (C). statistical difference in expression in different comparison groups, and (D). Wayne diagram of different comparison groups and total number of differential genes in different comparison groups.

Figure 4

(A). KEGG enrichment analysis, (B). transcription factor family analysis, (C). differential gene cluster analysis, and (D). WGCNA modular identification.

Figure 5

The expression levels of eight key differentially expressed genes in MF and MS plants were analyzed by qRT-PCR and RNA-Seq. The FPKM value is based on RNA-Seq data. The qRT-PCR was repeated three times, and the error bar indicated the standard error. The red stripe represents MS plants, and the blue stripe represents MF plants. ‘*’ indicated that the difference was statistically significant (t-test, p < 0.05), and ‘**’ indicated that the difference was extremely significant (p < 0.01).