Mechanism Of Male Sterility Research Articles

Lateral metabolome study reveals the molecular mechanism of cytoplasmic male sterility (CMS) in Chinese cabbage

BackgroundChinese cabbage is one of the most widely grown leafy vegetables in China. Cytoplasmic male sterility (CMS) is a maternally inherited trait that produces abnormal pollen during anther development, which is commonly seen in cruciferous vegetables. However, the molecular mechanism of Chinese cabbage CMS is not clear. In this study, the metabolome and hormone profiles of Chinese cabbage male sterile line (CCR20000) and sterile maintainer line (CCR20001) were analyzed in flower buds during normal stamen development and abnormal stamen development, respectively.ResultsA total of 556 metabolites were detected based on UPLC-MS/MS detection platform and database search, and the changes of hormones such as auxin, cytokinins, abscisic acid, jasmonates, salicylic acid, gibberellin acid and ethylene were analyzed. The results showed that compared with the male fertile line (MF), the male sterile line (MS) significantly decreased the content of flavonoids and phenolamides metabolites in the stamen dysplasia stage, accompanied by a large accumulation of glucosinolate metabolites. Meanwhile, the contents of GA9, GA20, IBA, tZ and other hormones in MS were significantly lower than those in MF strains. Further, by comparing the metabolome changes of MF and MS during stamen dysplasia, it was found that flavonoid metabolites and amino acid metabolites were distinctly different.ConclusionsThese results suggest that flavonoids, phenolamides and glucosinolate metabolites may be closely related to the sterility of MS strains. This study provides an effective basis for further research on the molecular mechanism of CMS in Chinese cabbage.

Integrated transcriptome and proteome analysis provides insights into the mechanism of cytoplasmic male sterility (CMS) in tobacco (Nicotiana tabacum L.)

Cytoplasmic male sterility (CMS) is critical in maximizing crop yield and quality by utilizing tobacco heterosis. However, the mechanism of tobacco CMS formation remains unknown. Using paraffin section observation, transcriptome sequencing, and TMT proteomic analysis, this study describes the differences in expression profiles in morphology, transcription, and translation between the sua-CMS tobacco line (MSYY87) and its corresponding maintainer line (YY87). According to the microspore morphology, MSYY87 began to exhibit abnormal microspore development during the early stages of germination and differentiation (androgynous primordium differentiation stage). According to transcriptomic and proteomic analyses, 17 genes/proteins involved in lipid transport/binding and phenylpropane metabolism were significantly down-regulated at both the mRNA and protein levels. Through further analysis, we identified some key genes that may be involved in tobacco male sterility, including β-GLU related to energy metabolism, 4CL and bHLHs related to anther wall formation, nsLTPs related to pollen germination and anther cuticle, and bHLHs related to pollen tapetum degradation. We speculate that the down-regulation of these genes affects the normal physiological metabolism, making tobacco plants show male sterility. SignificanceCytoplasmic male sterility (CMS) plays a vital role in utilizing tobacco heterosis and enhancing crop yield and quality. We observed paraffin sections and conducted transcriptome sequencing and mitochondrial proteomics to examine the tobacco CMS line Yunyan 87 (MSYY87) and its maintainer line Yunyan 87 (YY87). The down-regulation expression of β-GLU resulted in insufficient ATP supply, which resulted in disordered energy metabolism. The down-regulation expression of 4CL, nsLTPs and bHLHs may affect the formation of anther wall and anther cuticle, pollen germination, as well as the degradation of pollen tapetum. These various abnormal physiological processes, the male sterility of tobacco is finally caused. The findings shed light on the molecular mechanisms of tobacco CMS and serve as a model for fertility research in other flowering plants.

Integrated analysis of transcriptome and proteome changes related to the male-sterile mutant MS7-2 in wucai (Brassica Campestris L.)

Male sterility is a complex developmental process involving the differentiation of different tissues and the coordinated expression of genes. It is not only used to produce hybrids with effective utilization of heterosis but also an ideal tool to study plant development networks. Here, we carried out morphological and cytological observations on the flower buds of the previously studied male-sterile mutant MS7-2 and its wild-type fertile plant MF7-2 at different developmental stages and performed transcriptomic and proteomic analyses. Compared with the anthers of MF7-2, the anthers of MS7-2 were obviously atrophied, the anther surface was concave, the tapetum was abnormally expanded at the mononuclear microspore stage and normal pollen grains could not be produced. Proteomic analysis showed that the differentially expressed proteins (DEPs) were mainly involved in lipid transport, lipid binding and lipase activity and were mainly concentrated in carbohydrate metabolism, biosynthesis of other secondary metabolites and lipid metabolism. Transcriptomic results showed that the differentially expressed genes (DEGs) were mainly related to cell membrane components and were significantly enriched in carbohydrate metabolism, lipid metabolism, membrane transport and other pathways. In addition, transcription factors involved in anther and pollen development were also found to be significantly down-regulated in MS7-2, such as AMS, bHLH66, bHLH69 and WRKY 34. Weighted gene coexpression network analysis (WGCNA) screened two key modules of anther abortion in wucai. The DEGs in the two modules were mainly enriched in the “terpenoid backbone biosynthesis,” “pentose and glucuronate interconversions” and “cutin, suberine and wax biosynthesis” pathways, and three hub genes were screened as BraA02g028550.3C (LACS5), BraA01g016670.3C (VHA-G3) and BraA03g043400. 3C (AMS). Through transcriptomic and proteomic analyses, a regulatory network of MS7-2 male sterility of was constructed. It is speculated that the down-regulation of these genes and proteins in MS7-2 anthers inhibits the synthesis of some lipids and endogenous metabolites and accumulates a large amount of reactive oxygen species (ROS) in the tapetum, leading to the failure of normal PCD and the abortion of MS7-2. This study provides new insights into the molecular mechanism of male sterility in wucai.

Duplicate mutations of GhCYP450 lead to the production of ms5m6 male sterile line in cotton.

The duplicated male sterile genes ms5m6 in cotton were map-based cloned and validated by the virus-induced gene silencing assays. Duplicate mutations of the GhCYP450 gene encoding a cytochrome P450 protein are responsible for the male sterility in cotton. The utilization of male sterility in cotton plays a vital role in improving yield and fiber quality. A complete male sterile line (ms5ms6) has been extensively used to develop hybrid cotton worldwide. Using Zhongkang-A (ZK-A) developed by transferring Bt and ms5ms6 genes into the commercial cultivar Zhongmiansuo 12, the duplicate genes were map-based cloned and confirmed via the virus-induced gene silencing (VIGS) assays. The duplicate mutations of GhCYP450 genes encoding a cytochrome P450 protein were responsible for producing male sterility in ms5ms6 in cotton. Sequence alignment showed that GhCYP450-Dt in ZK-A differed in two critical aspects from the fertile wild-type TM-1: GhCYP450-Dt has three amino acid (D98E, E168K, G198R) changes in the coding region and a 7-bp (GGAAAAA) insertion in the promoter domain; GhCYP450-At appears to be premature termination of GhCYP450 translation. Further morphological observation and cytological examination of GhCYP450-silenced plants induced by VIGS exhibited shorter filaments and no mature pollen grains. These results indicate that GhCYP450 is essential for pollen exine formation and pollen development for male fertility. Investigating the mechanisms of ms5ms6 male sterility will deepen our understanding of the development and utilization of heterosis.

BrACOS5 mutations induced male sterility via impeding pollen exine formation in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

BrACOS5 mutations led to male sterility of Chinese cabbage verified in three allelic male-sterile mutants. Chinese cabbage (Brassica rapa L. ssp. pekinensis) is one of the major vegetable crops in East Asia, and the utilization of male-sterile line is an important measure for its hybrid seed production. Herein, we isolated three allelic male-sterile mutants, msm1-1, msm1-2 and msm1-3, from an ethyl methane sulfonate (EMS) mutagenized population of Chinese cabbage double-haploid (DH) line 'FT', whose microspores were completely aborted with severely absent exine, and tapetums were abnormally developed. Genetic analyses indicated that the three male-sterile mutants belonged to allelic mutation and were triggered by the same recessive nuclear gene. MutMap-based gene mapping and kompetitive allele-specific PCR (KASP) analysis demonstrated that three different single-nucleotide polymorphisms (SNPs) of BraA09g012710.3C were responsible for the male sterility of msm1-1/2/3, respectively. BraA09g012710.3C is orthologous of Arabidopsis thaliana ACOS5 (AT1G62940), encoding an acyl-CoA synthetase in sporopollenin biosynthesis, and specifically expressed in anther, so we named BraA09g012710.3C as BrACOS5. BrACOS5 localizes to the endoplasmic reticulum (ER). Mutations of BrACOS5 resulted in decreased enzyme activities and altered fatty acid contents in msm1 anthers. As well as the transcript accumulations of putative orthologs involved in sporopollenin biosynthesis were significantly down-regulated excluding BrPKSA. These results provide strong evidence for the integral role of BrACOS5 in conserved sporopollenin biosynthesis pathway and also contribute to uncovering exine development pattern and underlying male sterility mechanism in Chinese cabbage.

Environment-sensitive genic male sterility in rice and other plants.

Environment-sensitive genic male sterility is a type of male sterility that is affected by both genetic and environmental factors. Environment-sensitive genic male sterile lines are not only used in two-line hybrid breeding but are also good materials for studying plant-environment interactions. In this study we review the research progress on environment-sensitive genic male sterility in rice from the perspectives of epigenetic, transcriptional, posttranscriptional, posttranslationaland metabolic mechanisms as well as signal transduction processes. While significant progress has been made in the genetics, gene cloningand understanding of the molecular mechanisms of environment-sensitive genic male sterility in recent years, the relevant regulatory network is still poorly understood in rice. We therefore also review studies of environment-sensitive genic male sterility in Arabidopsis and other crops, hoping to promote research in this field in rice. Finally, we analyse the challenges posed by environment-sensitive genic male sterility and provide corresponding suggestions. This review will contribute towards an understanding the molecular genetics of environment-sensitive genic male sterility and its application in two-line hybrid breeding in rice and other species.

Genome-Wide Identification and Expression Analysis of TUA and TUB Genes in Wheat (Triticum aestivum L.) during Its Development.

Microtubules play a fundamental role in plant development, morphogenesis, and cytokinesis; they are assembled from heterodimers containing an α-tubulin (TUA) and a β-tubulin (TUB) protein. However, little research has been conducted on the TUA and TUB gene families in hexaploid wheat (Triticum aestivum L.). In this study, we identified 15 TaTUA and 28 TaTUB genes in wheat. Phylogenetic analysis showed that 15 TaTUA genes were divided into two major subfamilies, and 28 TaTUB genes were divided into five major subfamilies. Mostly, there were similar motif compositions and exon-intron structures among the same subfamilies. Segmental duplication of genes (WGD/segmental) is the main process of TaTUA and TaTUB gene family expansion in wheat. It was found that TaTUA and TaTUB genes presented specific temporal and spatial characteristics based on the expression profiles of 17 tissues during wheat development using publicly available RNA-seq data. It was worth noting, via qRT-PCR, that two TaTUA and five TaTUB genes were highly expressed in fertile anthers compared to male sterility. These were quite different between physiological male sterile lines and S-type cytoplasmic male sterile lines at different stages of pollen development. This study offers fundamental information on the TUA and TUB gene families during wheat development and provides new insights for exploring the molecular mechanism of wheat male sterility.

Epididymal epithelial degeneration and lipid metabolism impairment account for male infertility in occludin knockout mice.

Occludin (OCLN) is a tight junction protein and Ocln deletion mutation causes male infertility in mice. However, the role of OCLN in male reproductive system remains unknown. In this study, we used an interdisciplinary approach to elucidate the underlying mechanism of male infertility in related to OCLN function, including Ocln knockout mice as well as a combined omics analysis and immunofluorescent labelling. Our results showed that the epididymis of Ocln-null mice displayed a phenomenon resembling epididymal sperm granuloma, which occurred especially in the junctional region between caput and corpus epididymidis. Sperm motility and fertilisation capacity were also impaired in these Ocln-null mice, accompanied by enlarged tubules in the proximal regions and degeneration in the distal regions of epididymis. Cellular localization analysis showed that OCLN immunofluorescence was enriched only in the apical junction of epithelial principal cells in the proximal regions of epididymis. Integrative omics analysis revealed the downregulation of gene clusters enriched in acid secretion and fatty acid metabolism in the Ocln-null epididymis, especially the enzymes related to the unsaturated arachidonic acid pathway. The number of proton-pump V-ATPase-expression clear cells, a key player of luminal acidification in the epididymis, declined drastically from prepubertal age before sperm arrival but not in the early postnatal age. This was accompanied by programmed cell death of clear cells and increased pH in the epididymal fluid of OCLN-deficient mice. The lipidomics results showed significantly increased levels of specific DAGs conjugated to unsaturated fatty acids in the Ocln-mutant. Immunofluorescent labelling showed that the arachidonic acid converting enzyme PTGDS and phospholipase PLA2g12a were prominently altered in the principal cells and luminal contents of the Ocln-mutant epididymis. Whereas the carboxylate ester lipase CES1, originally enriched in the WT basal cells, was found upregulated in the Ocln-mutant principal cells. Overall, this study demonstrates that OCLN is essential for maintaining caput-to-corpus epithelial integrity, survival of acid-secreting clear cells, and unsaturated fatty acid catabolism in the mouse epididymis, thereby ensuring sperm maturation and male fertility.

Abnormal Programmed Cell Death of Tapetum Leads to the Pollen Abortion of Lycium barbarum Linnaeus

Programmed cell death (PCD) in tapetum provides nutrients for pollen development. Once this process becomes abnormal, the pollen will be aborted, and sterile lines will be formed. Lycium barbarum L. is a well-known medicinal and edible plant, and male sterile lines play an important role in the cultivation of its new varieties by utilizing hybrid vigor. However, the male sterility mechanism of L. barbarum is poorly understood. In this study, the main characteristic changes during the pollen development of L. barbarum sterile line (Ningqi No. 5) and fertile line (Ningqi No. 1) were compared through anatomical observation, physiological detection and gene expression analysis. Anatomical observation showed that compared with that of the fertile line, the tapetum of the sterile line persisted during pollen development, the organelle morphology and number of tapetal cells changed remarkably, and the PCD was remarkably delayed. Membranous peroxidation caused by reactive oxygen species (ROS) in the sterile line occurred from the late tetrad to the pollen grain stage, and that in Ningqi No. 1 transpired in the tetrad stage. This difference in the dynamic changes of ROS affected the redox balance of microspore development. qPCR detection of DYT1 and MS1 genes regulating tapetum development showed that compared with those in the fertile line, the expression levels of both genes in the sterile line changed significantly from pollen mother cell stage to pollen grain stage. This finding may be associated with the start-up delay of tapetal PCD. All these results suggested that abnormal tapetal PCD is an important mechanism leading to male sterility in L. barbarum.

The Role of Long Noncoding RNAs on Male Infertility: A Systematic Review and In Silico Analysis.

Simple SummaryMale infertility is a health problem affecting a huge number of couples worldwide. For many years, scientists have focused on protein-coding genes to explore the causes and mechanisms of male infertility. In addition, noncoding regions that are transcribed but do not lead to protein production have also been proven to play an important role in many cellular processes and diseases. The exact role of long noncoding RNAs on male infertility is not well understood. In order to address this gap, we performed a systematic review of the literature using two databases, and then a bioinformatics analysis to identify the most important long noncoding RNAs in male infertility, as well as their mechanism of action. At first, we identified 625 articles, but after selecting some of them according to the criteria that we defined, our final sample comprised 20 articles. After analyzing them, many long noncoding RNAs were found to be deregulated in male infertility or specific subtypes of it, paving the way for a better understanding of the molecular mechanisms behind male infertility, or even for the improvement of diagnosis and therapy. Finally, an association was found between male infertility and cancer that requires further investigation in the future.Male infertility is a complex disorder affecting many couples worldwide. Long noncoding RNAs (lncRNAs) regulate important cellular processes; however, a comprehensive understanding of their role in male infertility is limited. This systematic review investigates the differential expressions of lncRNAs in male infertility or variations in lncRNA regions associated with it. The PRISMA guidelines were used to search Pubmed and Web of Science (1 June 2022). Inclusion criteria were human participants, patients diagnosed with male infertility, and English language speakers. We also performed an in silico analysis investigating lncRNAs that are reported in many subtypes of male infertility. A total of 625 articles were found, and after the screening and eligibility stages, 20 studies were included in the final sample. Many lncRNAs are deregulated in male infertility, and interactions between lncRNAs and miRNAs play an important role. However, there is a knowledge gap regarding the impact of variants found in lncRNA regions. Furthermore, eight lncRNAs were identified as differentially expressed in many subtypes of male infertility. After in silico analysis, gene ontology (GO) and KEGG enrichment analysis of the genes targeted by them revealed their association with bladder and prostate cancer. However, pathways involved in general in tumorigenesis and cancer development of all types, such as p53 pathways, apoptosis, and cell death, were also enriched, indicating a link between cancer and male infertility. This evidence, however, is preliminary. Future research is needed to explore the exact mechanism of action of the identified lncRNAs and investigate the association between male infertility and cancer.

MicroRNA-34b-5p targets PPP1R11 to inhibit proliferation and promote apoptosis in cattleyak Sertoli cells by regulating specific signaling pathways

Cattleyaks, a hybrid of (♂) and yak (♀), exhibit the marked productivity and adaptability of plateau, but suffer from male infertility. Small non-coding RNAs, especially miRNAs, play crucial roles in spermatogenesis and affect the growth of Sertoli cells (SCs). The objective of the present study was to explore the interaction between miR-34b-5p and protein phosphatase 1 regulatory inhibitor subunit 11 (PPP1R11) and its effect on cattleyak SCs. RT-qPCR was used to determine the expression pattern of miR-34b-5p and PPP1R11, while the cellular and subcellular localization of PPP1R11 was determined by immunohistochemistry and immunocytochemistry. The interaction between MiR-34b-5p and PPP1R11 was evaluated by immunofluorescence, proliferation, apoptosis, and western blotting assays. The potential binding sites between miR-34b-5p and PPP1R11 were uncovered through targeted search of an online database, and verified using a dual luciferase reporter system. Our data show that miR-34b-5p is differentially expressed in the testes and SCs of cattleyaks compared to yaks. Overexpression of miR-34b-5p in SCs suppressed proliferation and induced apoptosis, while the effects of miR-34b-5p knockdown were the reverse. The 3′UTR of PPP1R11 was identified as a potential target site of miR-34b-5p, and this was validated by online database searches and our data from the dual-luciferase reporter assay, and it displayed an inverse expression pattern to miR-34b-5p in SCs. The effects of silencing PPP1R11 by siRNA were similar to the results of miR-34b-5p upregulation, but significantly different from miR-34b-5p downregulation in cattleyak SCs. The effects with PPP1R11 overexpression were opposite, suggesting a novel biofunctional role of PPP1R11 inactivation in depressing cattleyak SCs growth. Lastly, we confirmed that miR-34b-5p inhibited PPP1R11 expression and induced apoptosis by regulating proliferation- and apoptosis-related genes in SCs. Thus, miR-34b-5p regulates the apoptosis and proliferation of cattleyak SCs via targeting PPP1R11, which can provide an innovative direction for exploring the mechanism of cattleyak male sterility.

Mechanism and Utilization of Ogura Cytoplasmic Male Sterility in Cruciferae Crops.

Hybrid production using lines with cytoplasmic male sterility (CMS) has become an important way to utilize heterosis in vegetables. Ogura CMS, with the advantages of complete pollen abortion, ease of transfer and a progeny sterility rate reaching 100%, is widely used in cruciferous crop breeding. The mapping, cloning, mechanism and application of Ogura CMS and fertility restorer genes in Brassica napus, Brassica rapa, Brassica oleracea and other cruciferous crops are reviewed herein, and the existing problems and future research directions in the application of Ogura CMS are discussed.

Cloning and Expression Analysis of HAT1 and HDAC1 in the Testes of Mature Yaks and Their Sterile Hybrids.

Simple SummaryCattle-yak is the hybrid between male cattle (Bos taurus) and female yak (Bos grunniens). Male cattle-yak can not produce normal sperm. The mechanisms that underlie cattle-yak male sterility have not been elucidated. Histone acetylation is a common regulation mode that plays an important role in the development of gametes. The objective of this study was to explore the molecular mechanism of male sterility in yak hybrids based on histone acetyltransferase 1 (HAT1) and histone deacetylase 1 (HDAC1), two enzymes that regulate histone acetylation. The mRNA and protein expression levels of HAT1 in the testes of adult cattle-yaks were significantly lower than in adult yaks, and the protein expression levels of HDAC1 were significantly higher than in yaks. In addition, H3K9 acetylation levels in cattle-yak testes were significantly lower than in yaks. These results suggest that male sterility in cattle-yaks might be associated with decreased histone acetylation levels in the testes.The objective of this study was to explore the molecular mechanism of male sterility in yak hybrids based on HAT1 and HDAC1. Total RNA was extracted from the testes of adult yaks (n = 11) and sterile cattle-yaks (n = 11) followed by reverse transcription. The coding sequence (CDS) of yak HAT1 and HDAC1 were obtained by conventional polymerase chain reaction (PCR) and gene cloning. The testicular mRNA and protein levels of HAT1 and HDAC1 in yaks and cattle-yaks were detected by quantitative PCR (qPCR) and Western blotting, respectively, and the histone H3 lysine 9 (H3K9) histone acetylation level in the testes of yaks and cattle-yaks was assayed using enzyme linked immunosorbent assay (ELISA). The results showed that the CDS of HAT1 and HDAC1 were 1242 bp and 1449 bp in length, encoding 413 and 482 amino acids, respectively; yaks had a similar mRNA sequence as cattle in both genes. The testicular mRNA and protein levels of HAT1 of cattle-yaks were significantly lower than those of yaks, and the protein level of HDAC1 was significantly higher than that of yaks. ELISA showed that the acetylation level of testicular H3K9 was significantly lower in yak hybrids than that of yaks. The present results suggest that the decreased level of HAT1 and increased level of HDAC1 may result in the decreased H3K9 acetylation in cattle-yaks and might be associated with their sterility.

CRISPR/Cas9-Induced Mutagenesis of TMS5 Confers Thermosensitive Genic Male Sterility by Influencing Protein Expression in Rice (Oryza sativa L.)

The development of thermosensitive genic male sterile (TGMS) lines is the key to breeding two-line hybrid rice, which has been widely applied in China to increase grain yield. CRISPR/Cas9 has been widely used in genome editing to create novel mutants in rice. In the present study, a super grain quality line, GXU 47, was used to generate a new TGMS line with specific mutations in a major TGMS gene tms5 generated with CRISPR/Cas9-mediated genome editing in order to improve the rice quality of two-line hybrids. A mutagenesis efficiency level of 75% was achieved, and three homozygous T-DNA-free mutant lines were screened out. The mutants exhibited excellent thermosensitive male fertility transformation characteristics with complete male sterility at ≥24 °C and desirable male fertility at around 21 °C. Proteomic analysis based on isobaric tags for relative and absolute quantification (iTRAQ) was performed to unveil the subsequent proteomic changes. A total of 192 differentially expressed proteins (DEPs), including 35 upregulated and 157 downregulated, were found. Gene ontology (GO) analysis revealed that the DEPs were involved in a single-organism biosynthetic process, a single-organism metabolic process, oxidoreductase activity, and catalytic activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEPs were involved in ubiquinone and other terpenoid quinone biosynthesis, the biosynthesis of secondary metabolites, metabolic pathways, and phenylpropanoid biosynthesis. Our study shows that high mutation efficiency was achieved in both target sites, and T-DNA-free mutant lines were obtained in the T1 generation. The present study results prove that it is feasible and efficient to generate an excellent mutant line with CRISPR/Cas9, which provides a novel molecular mechanism of male sterility caused by the mutation of tms5.

Conjunctive Analyses of BSA-Seq and BSR-Seq Unveil the Msβ-GAL and MsJMT as Key Candidate Genes for Cytoplasmic Male Sterility in Alfalfa (Medicago sativa L.).

Knowing the molecular mechanism of male sterility in alfalfa is important to utilize the heterosis more effectively. However, the molecular mechanisms of male sterility in alfalfa are still unclear. In this study, the bulked segregant analysis (BSA) and bulked segregant RNA-seq (BSR) were performed with F2 separation progeny to study the molecular mechanism of male sterility in alfalfa. The BSA-seq analysis was located in a candidate region on chromosome 5 containing 626 candidate genes which were associated with male sterility in alfalfa, while the BSR-seq analysis filtered seven candidate DEGs related to male sterility, and these candidate genes including EF-Tu, β-GAL, CESA, PHGDH, and JMT. The conjunctive analyses of BSR and BSA methods revealed that the genes of Msβ-GAL and MsJMT are the common detected candidate genes involved in male sterility in alfalfa. Our research provides a theory basis for further study of the molecular mechanism of male sterility in alfalfa and significant information for the genetic breeding of Medicago sativa.

Integrated Methylome and Transcriptome Analysis Provides Insights into the DNA Methylation Underlying the Mechanism of Cytoplasmic Male Sterility in Kenaf (Hibiscus cannabinus L.).

Cytoplasmic male sterility (CMS) is widely exploited in hybrid seed production. Kenaf is an important fiber crop with high heterosis. The molecular mechanism of kenaf CMS remains unclear, particularly in terms of DNA methylation. Here, using the anthers of a kenaf CMS line (P3A) and its maintainer line (P3B), comparative physiological, DNA methylation, and transcriptome analyses were performed. The results showed that P3A had considerably lower levels of IAA, ABA, photosynthetic products and ATP contents than P3B. DNA methylome analysis revealed 650 differentially methylated genes (DMGs) with 313 up- and 337 down methylated, and transcriptome analysis revealed 1788 differentially expressed genes (DEGs) with 558 up- and 1230 downregulated genes in P3A compared with P3B. Moreover, 45 genes were characterized as both DEGs and DMGs, including AUX, CYP, BGL3B, SUS6, AGL30 and MYB21. Many DEGs may be regulated by related DMGs based on methylome and transcriptome studies. These DEGs were involved in carbon metabolism, plant hormone signal transduction, the TCA cycle and the MAPK signaling pathway and were shown to be important for CMS in kenaf. These results provide new insights into the epigenetic mechanism of CMS in kenaf and other crops.

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.

Transcriptome profiling of flower buds of male-sterile lines provides new insights into male sterility mechanism in alfalfa

BackgroundThe use of heterosis to produce hybrid seeds is a challenge to breeding for improved crop yield. In previous studies, we isolated a male sterile alfalfa hybrid and successfully obtained a genetically stable alfalfa male sterile line through backcrossing, henceforth named MS-4. In this study, we used RNA-seq technology to analyze the transcriptome profiles of the male sterile line (MS-4) and the male fertile line (MF) of alfalfa to elucidate the mechanism of male sterility.ResultsWe screened a total of 11,812 differentially expressed genes (DEGs) from both MS-4 and MF lines at three different stages of anther development. Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that these DEGs are mainly involved in processes such as energy metabolism, lipid and amino acid metabolism, carbohydrate metabolism, in addition to cell synthesis and aging. The results from protein–protein interaction (PPI) network analysis showed that the ribosomal protein (MS.Gene25178) was the core gene in the network. We also found that transcriptional regulation was an influential factor in the development of anthers.ConclusionsOur findings provide new insights into understanding of the fertility changes in the male sterile (MS-4) of alfalfa.

The chimeric gene atp6c confers cytoplasmic male sterility in maize by impairing the assembly of the mitochondrial ATP synthase complex

Cytoplasmic male sterility (CMS) is a powerful tool for the exploitation of hybrid heterosis and the study of signaling and interactions between the nucleus and the cytoplasm. C-type CMS (CMS-C) in maize has long been used in hybrid seed production, but the underlying sterility factor and its mechanism of action remain unclear. In this study, we demonstrate that the mitochondrial gene atp6c confers male sterility in CMS-C maize. The ATP6C protein shows stronger interactions with ATP8 and ATP9 than ATP6 during the assembly of F1Fo-ATP synthase (F-type ATP synthase, ATPase), thereby reducing the quantity and activity of assembled F1Fo-ATP synthase. By contrast, the quantity and activity of the F1' component are increased in CMS-C lines. Reduced F1Fo-ATP synthase activity causes accumulation of excess protons in the inner membrane space of the mitochondria, triggering a burst of reactive oxygen species (ROS), premature programmed cell death of the tapetal cells, and pollen abortion. Collectively, our study identifies a chimeric mitochondrial gene (ATP6C) that causes CMS in maize and documents the contribution of ATP6C to F1Fo-ATP synthase assembly, thereby providing novel insights into the molecular mechanisms of male sterility in plants.

Electrophysiological, Morphologic, and Transcriptomic Profiling of the Ogura-CMS, DGMS and Maintainer Broccoli Lines.

To better serve breeding of broccoli, the electrophysiological, morphological and transcriptomic profiling of the isogenic Ogura-CMS, DGMS and their maintainer fertile lines, were carried out by scanning electron microscopy, investigation of agronomic traits and RNA-sequencing analysis. The agronomic traits of plant height, length of the largest leaf, plant spread angle, single head weight, head width and stem diameter showed stronger performance in Ogura-CMS broccoli than in DGMS line or maintainer fertile line. However, the Ogura-CMS broccoli was poorer in the seed yield and seed germination than in the DGMS line and maintainer fertile line. Additionally, the DGMS broccoli had longer maturation and flowering periods than the Ogura-CMS and maintainer fertile lines. There were obvious differences in the honey gland, happening in the male sterility and fertile lines of broccoli. Additionally, the mechanism regulating Ogura-CMS and DGMS in broccoli was investigated using florets transcriptome analyses of the Ogura-CMS, DGMS and maintainer fertile lines. As a result, a total of 2670 differentially expressed genes (DEGs) were detected, including 1054 up- and 1616 downregulated genes in the Ogura-CMS and DGMS lines compared to the maintainer fertile line. A number of functionally known genes involved in plant hormones (auxin, salicylic acid and brassinosteroid), five Mitochondrial Oxidative Phosphorylation (OXPHOS) genes of atp8, LOC106319879, LOC106324734, LOC106314622 and LOC106298585, and three upregulated genes (Lhcb1, Lhcb3 and Lhcb5) associated with the photosynthesis-antenna protein pathway, were obviously detected to be highly associated with reproductive development including flowering time, maturity and reproductive period in the Ogura-CMS and DGMS broccoli comparing to their maintainer fertile line. Our research would provide a comprehensive foundation for understanding the differences of electrophysiological, morphological and transcriptomic profiles in the Ogura-CMS, DGMS and maintainer broccoli, and as well as being beneficial to exploring the mechanism of male sterility in Brassica crops.