Xiyue Song

Cytochrome c plays a central role in mitochondrial electron transport and reactive oxygen species (ROS) homeostasis, yet the function of cytochrome c-encoding genes in thermo-sensitive male sterility in wheat remains largely unexplored. In this study, we cloned and characterized TaCytc, an anther-preferentially expressed cytochrome c gene, from KTM3315A, a wheat line exhibiting thermo-sensitive male sterility with Aegilops kotschyi cytoplasm (K-TCMS). TaCytc encodes a protein containing a conserved Cyc7 domain and localizes to the mitochondrial membrane. Expression analysis revealed that TaCytc transcripts peaked at the binucleate stage in fertile anthers and were significantly downregulated under sterile conditions. Virus-induced gene silencing of TaCytc under fertile conditions resulted in complete male sterility, accompanied by impaired anther dehiscence, pollen abortion, and a marked reduction in seed setting rate. Mechanistically, TaCytc silencing led to excessive ROS accumulation, reduced activities of antioxidant enzymes (SOD, POD, and CAT), and altered expression of ROS biosynthesis- and scavenging-related genes. Furthermore, we identified the transcription factor TaFUS as a direct upstream activator of TaCytc, which binds to its promoter and positively regulates its transcription. Collectively, our findings demonstrate that TaCytc maintains anther redox homeostasis by modulating ROS metabolism, thereby acting as a key determinant of fertility conversion in KTM3315A. The TaFUS–TaCytc regulatory axis provides a novel molecular link between nuclear transcriptional control and mitochondrial function in thermo-sensitive male sterility, with potential implications for hybrid wheat breeding.

Background: Photo-thermo-sensitive male sterility (PTMS), which refers to the male sterility triggered by variations in photoperiod and temperature, is a crucial element in the wheat two-line hybrid system. The development of safe production and efficient propagation for male sterile lines holds utmost importance in two-line hybrid wheat. Under the stable photoperiod condition, PTMS is mainly induced by high or low temperatures in wheat, but the effect of daily temperature difference (DTD) on the fertility conversion of PTMS lines has not been reported. Here, three BS type PTMS lines including BS108, BS138, and BS366, as well as a control wheat variety J411 were used to analyze the correlation between fertility and DTD using differentially sowing tests, photo-thermo-control experiments, and transcriptome sequencing. Results: The differentially sowing tests suggested that the optimal sowing time for safe seed production of the three PTMS lines was from October 5th to 25th in Dengzhou, China. Under the condition of 12 h 12 °C, the PTMS lines were greatly affected by DTD and exhibited complete male sterility at a temperature difference of 15 °C. Furthermore, under different temperature difference conditions, a total of 20,677 differentially expressed genes (DEGs) were obtained using RNA sequencing. Moreover, through weighted gene co-expression network analysis (WGCNA) and KEGG enrichment analysis, the identified DEGs had a close association with “starch and sucrose metabolism”, “phenylpropanoid biosynthesis”, “MAPK signaling pathway-plant”, “flavonoid biosynthesis”, and “cutin, and suberine and wax biosynthesis”. qRT-PCR analysis showed the expression levels of core genes related to KEGG pathways significantly decreased at a temperature difference of 15 ° C. Finally, we constructed a transcriptome mediated network of temperature difference affecting male sterility. Conclusions: The findings provide important theoretical insights into the correlation between temperature difference and male sterility, providing guidance for the identification and selection of more secure and effective PTMS lines.

Main conclusion TaAGL66, a MADS-box transcription factor highly expressed in fertile anthers of KTM3315A, regulates anther and/or pollen development, as well as male fertility in wheat with Aegilops kotschyi cytoplasm. Abstract Male sterility, as a string of sophisticated biological processes in higher plants, is commonly regulated by transcription factors (TFs). Among them, MADS-box TFs are mainly participated in the processes of floral organ formation and pollen development, which are tightly related to male sterility, but they have been little studied in the reproductive development in wheat. In our study, TaAGL66, a gene that was specifically expressed in spikes and highly expressed in fertile anthers, was identified by RNA sequencing and the expression profiles data of these genes, and qRT-PCR analyses, which was localized to the nucleus. Silencing of TaAGL66 under fertility condition in KTM3315A, a thermo-sensitive male sterile line with Ae. kotschyi cytoplasm, displayed severe fertility reduction, abnormal anther dehiscence, defective pollen development, decreased viability, and low seed-setting. It can be concluded that TaAGL66 plays an important role in wheat pollen development in the presence of Ae. kotschyi cytoplasm, providing new insights into the utilization of male sterility.

Main conclusion The loss of TaMYB305 function down-regulated the expression of jasmonic acid synthesis pathway genes, which may disturb the jasmonic acid synthesis, resulting in abnormal pollen development and reduced fertility. Abstract The MYB family, as one of the largest transcription factor families found in plants, regulates plant development, especially the development of anthers. Therefore, it is important to identify potential MYB transcription factors associated with pollen development and to study its role in pollen development. Here, the transcripts of an R2R3 MYB gene TaMYB305 from KTM3315A, a thermo-sensitive cytoplasmic male-sterility line with Aegilops kotschyi cytoplasm (K-TCMS) wheat, was isolated. Quantitative real-time PCR (qRT-PCR) and promoter activity analysis revealed that TaMYB305 was primarily expressed in anthers. The TaMYB305 protein was localized in the nucleus, as determined by subcellular localization analysis. Our data demonstrated that silencing of TaMYB305 was related to abnormal development of stamen, including anther indehiscence and pollen abortion in KAM3315A plants. In addition, TaMYB305-silenced plants exhibited alterations in the transcriptional levels of genes involved in the synthesis of jasmonic acid (JA), indicating that TaMYB305 may regulate the expression of genes related to JA synthesis and play an important role during anther and pollen development of KTM3315A. These results provide novel insight into the function and molecular mechanism of R2R3-MYB genes in pollen development.

Key Message QMS-5B, a major QTL for photo-thermo-sensitive genic male sterility in wheat, was fine mapped in a 2.15 Mb region harboring a serine/threonine protein kinase gene TraesCS5B03G0887500, which was the most likely candidate gene. Abstract Genic male sterility is an essential trait in the utilization of heterosis and hybrid seed production for wheat. Currently, genic male sterile genes have been reported in wheat mutants, but the sterile genes controlling photo-thermo-sensitive genic male sterility in wheat have not been studied systematically. Here, 235 doubled haploid lines derived from a cross between photo-thermo-sensitive genic male sterile line BS462 and its restorer line CP279 were used to map male sterile gene by GenoBaits® Wheat 100 K Panel, bulked segregant exome sequencing (BSE-Seq) and wheat 660 K array. As a result, the major stable QTL on chromosome 5B, QMS-5B, was identified in all four environments, accounting for 7.3–36.4% of the phenotypic variances. Ulteriorly, QMS-5B was delimited to an approximate 2.15 Mb physical interval between KASP-5B5 and KASP-5B6 using kompetitive allele-specific PCR (KASP) markers. Within the interval, twenty-nine high-confidence genes were predicted according to Chinese Spring RefSeq v2.1. TraesCS5B03G0887500, encoding a serine/threonine protein kinase, was identified as the most likely candidate gene for QMS-5B based on weighted gene co-expression network analysis. Expression analysis confirmed that TraesCS5B03G0887500 was significantly differentially expressed in anthers of BS462 and CP279 at different stages under fertile and sterile environments. In addition, flanking KASP marker KASP-5B6 can effectively genotype male sterile lines and restorer lines, and can be used for molecular marker-assisted selection. This study provides insights into for exploring the mechanism of photo-thermo-sensitive genic male sterility in wheat.

Summary: High‐molecular‐weight glutenin subunits (HMW‐GS), a major component of seed storage proteins (SSP) in wheat, largely determine processing quality. HMW‐GS encoded by GLU‐1 loci are mainly controlled at the transcriptional level by interactions between cis‐elements and transcription factors (TFs). We previously identified a conserved cis‐regulatory module CCRM1‐1 as the most essential cis‐element for Glu‐1 endosperm‐specific high expression. However, the TFs targeting CCRM1‐1 remained unknown. Here, we built the first DNA pull‐down plus liquid chromatography‐mass spectrometry platform in wheat and identified 31 TFs interacting with CCRM1‐1. TaB3‐2A1 as proof of concept was confirmed to bind to CCRM1‐1 by yeast one hybrid and electrophoretic mobility shift assays. Transactivation experiments demonstrated that TaB3‐2A1 repressed CCRM1‐1‐driven transcription activity. TaB3‐2A1 overexpression significantly reduced HMW‐GS and other SSP, but enhanced starch content. Transcriptome analyses confirmed that enhanced expression of TaB3‐2A1 down‐regulated SSP genes and up‐regulated starch synthesis‐related genes, such as TaAGPL3, TaAGPS2, TaGBSSI, TaSUS1 and TaSUS5, suggesting that it is an integrator modulating the balance of carbon and nitrogen metabolism. TaB3‐2A1 also had significant effects on agronomic traits, including heading date, plant height and grain weight. We identified two major haplotypes of TaB3‐2A1 and found that TaB3‐2A1‐Hap1 conferred lower seed protein content, but higher starch content, plant height and grain weight than TaB3‐2A1‐Hap2 and was subjected to positive selection in a panel of elite wheat cultivars. These findings provide a high‐efficiency tool to detect TFs binding to targeted promoters, considerable gene resources for dissecting regulatory mechanisms underlying Glu‐1 expression, and a useful gene for wheat improvement.