The R2R3 MYB gene TaMYB305 positively regulates anther and pollen development in thermo-sensitive male-sterility wheat with Aegilops kotschyi cytoplasm.

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. 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.

The transcription factor TaGAMYB modulates tapetum and pollen development of TGMS wheat YanZhan 4110S via the gibberellin signaling.

Male reproductive development in higher plants experienced a series of complex biological processes, which can be regulated by Gibberellins (GA). The transcriptional factor GAMYB is a crucial component of GA signaling in anther development. However, the mechanism of GAMYB in wheat male reproduction is less understood. Here, we found that the thermo-sensitive genic male sterilitywheat line YanZhan 4110S displayed delayed tapetum programmed cell death and pollen abortive under the hot temperature stress. Combined with RNA-Sequencing data analysis, TaGAMYB associated with fertility conversion was isolated, which was located in the nucleus and highly expressed in fertility anthers. The silencing of TaGAMYB in wheat displayed fertility decline, defects in tapetum, pollen and exine formation, where the abortion characteristics were the same as YanZhan 4110S. In addition, either hot temperature or GA3 treatment in YanZhan 4110S caused the downregulation of TaGAMYB at binucleate stage and trinucleate stage, as well as fertility decrease. Further, the transcription factor TaWRKY2 significantly changed under GA3-treatment and directly interacted with the TaGAMYB promoter by W-box cis-element. Therefore, we suggested that TaGAMYB may be essential for anther development and male fertility, and GA3 activates TaGAMYB by TaWRKY2 to regulate fertility in wheat.

TaEXPB5 functions as a gene related to pollen development in thermo-sensitive male-sterility wheat with Aegilops kotschyi cytoplasm.

The thermo-sensitive cytoplasmic male-sterility line with Aegilops kotschyi cytoplasm (K-TCMS) is completely male sterile under low temperature (< 18 ℃) during Zadoks growth stages 45-52, whereas its fertility can be restored under hot temperature (≥ 20 ℃). The K-TCMS line may facilitate hybrid breeding and hybrid wheat production. Therefore, to elucidate the molecular mechanisms of its male sterility/fertility conversion, we conducted the association analysis of proteins and transcript expression to screen fertility related genes using RNA-seq, iTRAQ, and PRM-based assay. A gene encoding expansin protein in wheat, TaEXPB5, was isolated in K-TCMS line KTM3315A, which upregulated expression in the fertility anthers. Subcellular localization analysis suggested that TaEXPB5 protein localized to nucleus and cell wall. The silencing of TaEXPB5 displayed pollen abortion and the declination of fertility. Further, cytological investigation indicated that the silencing of TaEXPB5 induced the early degradation of tapetum and abnormal development of pollen wall. These results implied that TaEXPB5 may be essential for anther or pollen development and male fertility of KTM3315A. These findings provide a novel insight into molecular mechanism of fertility conversion for thermo-sensitive cytoplasmic male-sterility wheat, and contribute to the molecular breeding of hybrid wheat in the future.

Genome Wide Identification and Characterization of Wheat GH9 Genes Reveals Their Roles in Pollen Development and Anther Dehiscence.

Glycoside hydrolase family 9 (GH9) is a key member of the hydrolase family in the process of cellulose synthesis and hydrolysis, playing important roles in plant growth and development. In this study, we investigated the phenotypic characteristics and gene expression involved in pollen fertility conversion and anther dehiscence from a genomewide level. In total, 74 wheat GH9 genes (TaGH9s) were identified, which were classified into Class A, Class B and Class C and unevenly distributed on chromosomes. We also investigated the gene duplication and reveled that fragments and tandem repeats contributed to the amplification of TaGH9s. TaGH9s had abundant hormone-responsive elements and light-responsive elements, involving JA-ABA crosstalk to regulate anther development. Ten TaGH9s, which highly expressed stamen tissue, were selected to further validate their function in pollen fertility conversion and anther dehiscence. Based on the cell phenotype and the results of the scanning electron microscope at the anther dehiscence period, we found that seven TaGH9s may target miRNAs, including some known miRNAs (miR164 and miR398), regulate the level of cellulose by light and phytohormone and play important roles in pollen fertility and anther dehiscence. Finally, we proposed a hypothesis model to reveal the regulation pathway of TaGH9 on fertility conversion and anther dehiscence. Our study provides valuable insights into the GH9 family in explaining the male sterility mechanism of the wheat photo-thermo-sensitive genetic male sterile (PTGMS) line and generates useful male sterile resources for improving wheat hybrid breeding.

The gene TaPG encoding a polygalacturonase is critical for pollen development and male fertility in thermo-sensitive cytoplasmic male-sterility wheat.

Thermo-sensitive cytoplasmic male sterility is of great significance to heterosis and hybrid seed production in wheat. Consequently, it is worthwhile to research the genes associated with male sterility. Although polygalacturonases (PGs) have been studied to play a crucial role in male reproduction of many plants, their functions in the reproductive development of wheat remain unclear. Here, TaPG (TraesCS7A02G404900) encoding a polygalacturonase was isolated from the anthers of KTM3315A, a wheat thermo-sensitive cytoplasmic male sterile with Aegilops kotschyi cytoplasm. Expression pattern analyses showed that TaPG was strongly expressed in fertile anthers and its protein was localized in the cell wall. Further verification via barley stripe mosaic virus revealed that the silencing of TaPG exhibited abnormal anthers, premature degradation of tapetum, pollen abortion, and defective pollen wall formation, resulting in the declination of fertility. Conclusively, our research suggested that TaPG contributed to the pollen development and male fertility, which will provide a novel insight into the fertility conversion of thermo-sensitive cytoplasmic male sterility in wheat.

Comprehensive analysis of LIM gene family in wheat reveals the involvement of TaLIM2 in pollen development.

LIM domain proteins were involved in organizing the cytoskeleton, adjusting the metabolism and gene expression, some of them were specific express in pollen. LIM gene family in plants were studied in sunflower, tobacco, foxtail millet, rape, rice and Arabidopsis thaliana, however, it has not been investigated in wheat to date. In the present study, we totally characterized 29 TaLIM genes through genome-wide analysis, which were divided into two categories and five subclasses according to phylogenetic analysis. RNA-Seq analysis indicated the expression patterns of TaLIM genes have specific temporal and spatial characteristics, especially TaLIM2 was highly expressed in fertility anthers. Phenotypic and cytological of BSMV: TaLIM2 showed that it had defects in the later stage of pollen development and germination, which further testified that TaLIM2 was closely related to fertility conversion. These findings will be useful for functional analysis of LIM genes in wheat fertility and contribute to hybrid wheat breeding.

Identification and verification of genes related to pollen development and male sterility induced by high temperature in the thermo-sensitive genic male sterile wheat line.

MAIN CONCLUSION: Bioinformatic analysis identified the function of genes regulating wheat fertility. Barley stripe mosaic virus-induced gene silencing verified that the genes TaMut11 and TaSF3 are involved in pollen development and related to fertility conversion. Environment-sensitive genic male sterility is of vital importance to hybrid vigor in crop production and breeding. Therefore, it is meaningful to study the function of the genes related to pollen development and male sterility, which is still not fully understand currently. In this study, YanZhan 4110S, a new thermo-sensitive genic male sterility wheat line, and its near-isogenic line YanZhan 4110 were analyzed. Through comparative transcriptome basic bioinformatics and weighted gene co-expression network to further identify some hub genes, the genes TaMut11 and TaSF3 associated with pollen development and male sterility induced by high-temperature were identified in YanZhan 4110S. Further verification through barley stripe mosaic virus-induced gene silencing elucidated that the silencing of TaMut11 and TaSF3 caused pollen abortion, finally resulting in the declination of fertility. These findings provided data on the abortive mechanism in environment-sensitive genic male sterility wheat.

Comprehensive analysis of polygalacturonase gene family highlights candidate genes related to pollen development and male fertility in wheat (Triticum aestivum L.).

MAIN CONCLUSION: Four polygalacturonase gene family members were highlighted that contribute to elucidate the roles of polygalacturonase during the fertility conversion process in male-sterile wheat. Polygalacturonase (PG) belongs to a large family of hydrolases with important functions in cell separation during plant growth and development via the degradation of pectin. Specific expressed PGs in anthers may be significant for male sterility research and hybrid wheat breeding, but they have not been characterized in wheat (Triticum aestivum L.). In this study, we systematically studied the PG gene family using the latest published wheat reference genomic information. In total, 113 wheat PG genes were identified, which could be classified into six categories A-F according to their structure characteristics and phylogenetic comparisons with Arabidopsis and rice. Polyploidy and segmental duplications in wheat were proved to be mainly responsible for the expansion of the wheat PG gene family. RNA-seq showed that TaPGs have specific temporal and spatial expression characteristics, in which 12 TaPGs with spike-specific expression patterns were detected by qRT-PCR in different fertility anthers of KTM3315A, a thermo-sensitive cytoplasmic male-sterile wheat. Four of them specific upregulated (TaPG09, TaPG95, and TaPG93) or downregulated (TaPG87) at trinucleate stage of fertile anthers, and further aligning with the homologous in Arabidopsis revealed that they may undertake functions such as anther dehiscence, separation of pollen, pollen development, and pollen tube elongation, thereby inducing male fertility conversion in KTM3315A. These findings facilitate function investigations of the wheat PG gene family and provide new insights into the fertility conversion mechanism in male-sterile wheat.

Comparative transcriptome analysis indicates conversion of stamens into pistil-like structures in male sterile wheat (Triticum aestivum L.) with Aegilops crassa cytoplasm.

BACKGROUND: Aegilops crassa cytoplasm is an important source for investigating cytoplasmic male sterility (CMS). Moreover, the stamens of line C303A exhibit a high degree of pistillody, turning almost white. However, the molecular mechanism that underlies pistillody in C303A remains unclear. Therefore, to obtain a better understanding of pistillody in C303A, the phenotypic and cytological features of C303A were observed to identify the key stage for the homeotic transformation of stamens into pistil-like structures. Transcriptome profiles were determined for stamens using Illumina RNA sequencing. RESULTS: Morphological observations of the CMS wheat line with Aegilops crassa cytoplasm C303A showed that the pistils developed normally, but the stamens were ultimately aborted and they released no pollen when mature. According to paraffin section observations, the stamens began to transform into pistils or pistil-like structures in the binucleate stage (BNS). Therefore, the stamens were collected from line C303A and its maintainer 303B in the BNS for transcriptome sequencing. In total, 20,444 wheat genes were determined as differentially expressed in C303A and 303B stamens, with 10,283 upregulated and 10,161 downregulated genes. Gene Ontology enrichment analyses showed that most of the differentially expressed genes (DEGs) were annotated with GO terms comprising metabolic process, cell, cellular process, catalytic activity, and cell part. Analysis based on the Kyoto Encyclopedia of Genes and Genomes database showed that the enriched DEGs were mainly associated with energy metabolism. We also found several essential genes that may contribute to pistillody in C303A. These findings suggest that disrupted energy metabolism and reactive oxygen metabolism induce pistillody and eventually lead to abortion in C303A. CONCLUSION: We determined the complex transcriptome profiles for C303A stamens and demonstrated that disrupted energy metabolism and class B MADS-box genes are related to pistillody. These findings may facilitate future studies of the mechanistic response of the wheat stamen and pollen development in CMS.

Comparative transcriptome analysis indicates that a core transcriptional network mediates isonuclear alloplasmic male sterility in wheat (Triticum aestivum L.).

BACKGROUND: Cytoplasmic male sterility (CMS) plays a crucial role in the utilization of heterosis and various types of CMS often have different abortion mechanisms. Therefore, it is important to understand the molecular mechanisms related to anther abortion in wheat, which remain unclear at present. RESULTS: In this study, five isonuclear alloplasmic male sterile lines (IAMSLs) and their maintainer were investigated. Cytological analysis indicated that the abortion type was identical in IAMSLs, typical and stainable abortion, and the key abortive period was in the binucleate stage. Most of the 1,281 core shared differentially expressed genes identified by transcriptome sequencing compared with the maintainer in the vital abortive stage were involved in the metabolism of sugars, oxidative phosphorylation, phenylpropane biosynthesis, and phosphatidylinositol signaling, and they were downregulated in the IAMSLs. Key candidate genes encoding chalcone--flavonone isomerase, pectinesterase, and UDP-glucose pyrophosphorylase were screened and identified. Moreover, further verification elucidated that due to the impact of downregulated genes in these pathways, the male sterile anthers were deficient in sugar and energy, with excessive accumulations of ROS, blocked sporopollenin synthesis, and abnormal tapetum degradation. CONCLUSIONS: Through comparative transcriptome analysis, an intriguing core transcriptome-mediated male-sterility network was proposed and constructed for wheat and inferred that the downregulation of genes in important pathways may ultimately stunt the formation of the pollen outer wall in IAMSLs. These findings provide insights for predicting the functions of the candidate genes, and the comprehensive analysis of our results was helpful for studying the abortive interaction mechanism in CMS wheat.

Blocked synthesis of sporopollenin and jasmonic acid leads to pollen wall defects and anther indehiscence in genic male sterile wheat line 4110S at high temperatures.

Environment-sensitive genic male sterility is a valid tool for hybrid production and hybrid breeding, but there are no previous reports of the molecular mechanism of fertility conversion. In this study, RNA-seq, phenotypic and cytological observations, and physiological indexes were applied to analyze thermo-sensitive genic male sterility line 4110S under different temperature conditions to explore the fertility transformation mechanism. In total, 3420 differentially expressed genes (DEGs) were identified comprising 2331 upregulated genes and 1089 downregulated genes. The DEGs were apparently distributed among 54 Gene Ontology functional groups. The phenylpropanoid, long-chain fatty acid, and jasmonic acid (JA) biosynthesis pathways were related to male sterility, where their downregulation blocked the synthesis of sporopollenin and JA. Phenotypic and cytological analyses showed that pollen wall defects and anther indehiscence at high temperatures induced sterility. Moreover, enzyme-linked immunosorbent assay results indicated that the abundance of JA was lower in 4110S under restrictive conditions (high temperature) than permissive conditions (low temperature). A possible regulated network of pathways associated with male sterility was suggested. These results provided insights into the molecular mechanism of fertility conversion in the thermosensitive male sterility system.

Tapetal-Delayed Programmed Cell Death (PCD) and Oxidative Stress-Induced Male Sterility of Aegilops uniaristata Cytoplasm in Wheat.

Cytoplasmic male sterility (CMS) plays a crucial role in the utilization of hybrid vigor. Pollen development is often accompanied by oxidative metabolism responses and tapetal programmed cell death (PCD), and deficiency in these processes could lead to male sterility. Aegilops uniaristata cytoplasmic male sterility (Mu-CMS) wheat is a novel male-sterile line in wheat, which possess important potential in hybrid wheat breeding. However, its CMS mechanisms remain poorly understood. In our study, U87B1-706A, with the Aegilops uniaristata cytoplasm, and the maintainer line 706B were used to explore the abortive reason. Compared with 706B, histological analysis and PCD detection of the anther demonstrated that U87B1-706A appeared as delayed tapetal PCD as well as a disorganized organelle phenotype in the early uninucleate stage. Subsequently, a shrunken microspore and disordered exine structure were exhibited in the late uninucleate stage. While the activities of antioxidase increased markedly, the nonenzymatic antioxidant contents declined obviously following overacummulation of reactive oxygen species (ROS) during pollen development in U87B1-706A. Real-time quantitative PCR testified that the transcript levels of the superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) genes, encoding pivotal antioxidant enzymes, were up-regulated in early pollen development. Therefore, we deduce excess ROS as a signal may be related to the increased expression levels of enzyme genes, thereby breaking the antioxidative system balance, resulting in delayed tapetal PCD initiation, which finally led to pollen abortion and male sterility in U87B1-706A. These results provide evidence to further explore the mechanisms of abortive pollen in CMS wheat.

iTRAQ-Based Proteomics Analyses of Sterile/Fertile Anthers from a Thermo-Sensitive Cytoplasmic Male-Sterile Wheat with Aegilops kotschyi Cytoplasm.

A “two-line hybrid system” was developed, previously based on thermo-sensitive cytoplasmic male sterility in Aegilops kotschyi (K-TCMS), which can be used in wheat breeding. The K-TCMS line exhibits complete male sterility and it can be used to produce hybrid wheat seeds during the normal wheat-growing season; it propagates via self-pollination at high temperatures. Isobaric tags for relative and absolute quantification-based quantitative proteome and bioinformatics analyses of the TCMS line KTM3315A were conducted under different fertility conditions to understand the mechanisms of fertility conversion in the pollen development stages. In total, 4639 proteins were identified, the differentially abundant proteins that increased/decreased in plants with differences in fertility were mainly involved with energy metabolism, starch and sucrose metabolism, phenylpropanoid biosynthesis, protein synthesis, translation, folding, and degradation. Compared with the sterile condition, many of the proteins that related to energy and phenylpropanoid metabolism increased during the anther development stage. Thus, we suggest that energy and phenylpropanoid metabolism pathways are important for fertility conversion in K-TCMS wheat. These findings provide valuable insights into the proteins involved with anther and pollen development, thereby, helping to further understand the mechanism of TCMS in wheat.