Combined Transcriptome and Proteome Analysis of Anthers of AL-type Cytoplasmic Male Sterile Line and Its Maintainer Line Reveals New Insights into Mechanism of Male Sterility in Common Wheat

Cytoplasmic male sterility (CMS) plays an essential role in hybrid seeds production. In wheat, orf279 was reported as a CMS gene of AL-type male sterile line (AL18A), but its sterility mechanism is still unclear. Therefore, transcriptomic and proteomic analyses of the anthers of AL18A and its maintainer line (AL18B) were performed to interpret the sterility mechanism. Results showed that the electron transport chain and ROS scavenging enzyme expression levels changed in the early stages of the anther development. Biological processes, i.e., fatty acid synthesis, lipid transport, and polysaccharide metabolism, were abnormal, resulting in pollen abortion in AL18A. In addition, we identified several critical regulatory genes related to anther development through combined analysis of transcriptome and proteome. Most of the genes were enzymes or transcription factors, and 63 were partially homologous to the reported genic male sterile (GMS) genes. This study provides a new perspective of the sterility mechanism of AL18A and lays a foundation to study the functional genes of anther development.

Disruption of the bHLH transcription factor Abnormal Tapetum 1 causes male sterility in watermelon

Although male sterility has been identified as a useful trait for hybrid vigor utilization and hybrid seed production, its underlying molecular mechanisms in Cucurbitaceae species are still largely unclear. Here, a spontaneous male-sterile watermelon mutant, Se18, was reported to have abnormal tapetum development, which resulted in completely aborted pollen grains. Map-based cloning demonstrated that the causal gene Citrullus lanatus Abnormal Tapetum 1 (ClATM1) encodes a basic helix-loop-helix (bHLH) transcription factor with a 10-bp deletion and produces a truncated protein without the bHLH interaction and functional (BIF) domain in Se18 plants. qRT–PCR and RNA in situ hybridization showed that ClATM1 is specifically expressed in the tapetum layer and in microsporocytes during stages 6–8a of anther development. The genetic function of ClATM1 in regulating anther development was verified by CRISPR/Cas9-mediated mutagenesis. Moreover, ClATM1 was significantly downregulated in the Se18 mutant, displaying a clear dose effect at the transcriptional level. Subsequent dual-luciferase reporter, β-glucuronidase (GUS) activity, and yeast one-hybrid assays indicated that ClATM1 could activate its own transcriptional expression through promoter binding. Collectively, ClATM1 is the first male sterility gene cloned from watermelon, and its self-regulatory activity provides new insights into the molecular mechanism underlying anther development in plants.

Transcriptomic analysis reveals candidate genes for male sterility in Prunus sibirica

Background The phenomenon of male sterility widely occurs in Prunus sibirica and has a serious negative impact on yield. We identified the key stage and cause of male sterility and found differentially expressed genes related to male sterility in Prunus sibirica, and we analyzed the expression pattern of these genes. This work aimed to provide valuable reference and theoretical basis for the study of reproductive development and the mechanisms of male sterility in Prunus sibirica. Method The microstructures of male sterile flower buds and male fertile flower buds were observed by paraffin section. Transcriptome sequencing was used to screen genes related to male sterility in Prunus sibirica. Quantitative real-time PCR analysis was performed to verify the transcriptome data. Results Anther development was divided into the sporogenous cell stage, tetrad stage, microspore stage, and pollen maturity stage. Compared with male fertile flower buds, in the microspore stage, the pollen sac wall tissue in the male sterile flower buds showed no signs of degeneration. In the pollen maturity stage, the tapetum and middle layer were not fully degraded, and anther development stopped. Therefore, the microspore stage was the key stage for anther abortion , and the pollen maturity stage was the post stage for anther abortion. A total of 4,108 differentially expressed genes were identified by transcriptome analysis. Among them, 1,899 were up-regulated, and 2,209 were down-regulated in the transcriptome of male sterile flower buds. We found that “protein kinase activity”, “apoptosis process”, “calcium binding”, “cell death”, “cytochrome c oxidase activity”, “aspartate peptidase activity”, “cysteine peptidase activity” and other biological pathways such as “starch and sucrose metabolism” and “proteasome” were closely related to male sterility in Prunus sibirica. A total of 331 key genes were preliminarily screened. Conclusion The occurrence of male sterility in Prunus sibirica involved many biological processes and metabolic pathways. According to the results of microstructure observations, related physiological indexes determination and transcriptome analysis, we reveal that the occurrence of male sterility in Prunus sibirica may be caused by abnormal metabolic processes such as the release of cytochrome c in the male sterile flower buds, the imbalance of the antioxidant system being destroyed, and the inability of macromolecular substances such as starch to be converted into soluble small molecules at the correct stage of reproductive development, resulting in energy loss. As a result, the tapetum cannot be fully degraded, thereby blocking anther development, which eventually led to the formation of male sterility.

A Novel R2R3-MYB Gene LoMYB33 From Lily Is Specifically Expressed in Anthers and Plays a Role in Pollen Development

Lily (Lilium spp.) is an important commercial flower crop, but its market popularity and applications are adversely affected by severe pollen pollution. Many studies have examined pollen development in model plants, but few studies have been conducted on flower crops such as lily. GAMYBs are a class of R2R3-MYB transcription factors and play important roles in plant development and biotic resistance; their functions vary in different pathways, and many of them are involved in anther development. However, their function and regulatory role in lily remain unclear. Here, the GAMYB homolog LoMYB33 was isolated and identified from lily. The open reading frame of LoMYB33 was 1620 bp and encoded a protein with 539 amino acids localized in the nucleus and cytoplasm. Protein sequence alignment showed that LoMYB33 contained a conserved R2R3 domain and three BOX motifs (BOX1, BOX2, and BOX3), which were unique to the GAMYB family. LoMYB33 had transcriptional activation activity, and its transactivation domain was located within 90 amino acids of the C-terminal. LoMYB33 was highly expressed during the late stages of anther development, especially in pollen. Analysis of the promoter activity of LoMYB33 in transgenic Arabidopsis revealed that the LoMYB33 promoter was highly activated in the pollen of stage 12 to 13 flowers. Overexpression of LoMYB33 in Arabidopsis significantly retarded growth; the excess accumulation of LoMYB33 also negatively affected normal anther development, which generated fewer pollen grains and resulted in partial male sterility in transgenic plants. Silencing of LoMYB33 in lily also greatly decreased the amount of pollen. Overall, our results suggested that LoMYB33 might play an important role in the anther development and pollen formation of lily.

Abnormal anther development leads to lower spikelet fertility in rice (Oryza sativa L.) under high temperature during the panicle initiation stage

Background Decreased spikelet fertility is often responsible for reduction in grain yield in rice (Oryza sativa L.). In this study, two varieties with different levels of heat tolerance, Liangyoupeijiu (LYPJ, heat susceptible) and Shanyou63 (SY63, heat tolerant) were subjected to two temperature treatments for 28 days during the panicle initiation stage in temperature/relative humidity-controlled greenhouses: high temperature (HT; 37/27 °C; day/night) and control temperature (CK; 31/27 °C; day/night) to investigate changes in anther development under HT during panicle initiation and their relationship with spikelet fertility. Results HT significantly decreased the grain yield of LYPJ by decreasing the number of spikelets per panicle and seed setting percentage. In addition, HT produced minor adverse effects in SY63. The decreased spikelet fertility was primarily attributed to decreased pollen viability and anther dehiscence, as well as poor pollen shedding of the anthers of LYPJ under HT. HT resulted in abnormal anther development (fewer vacuolated microspores, un-degraded tapetum, unevenly distributed Ubisch bodies) and malformation of pollen (obscure outline of the pollen exine with a collapsed bacula, disordered tectum, and no nexine of the pollen walls, uneven sporopollenin deposition on the surface of pollen grains) in LYPJ, which may have lowered pollen viability. Additionally, HT produced a compact knitted anther cuticle structure of the epidermis, an un-degraded septum, a thickened anther wall, unevenly distributed Ubisch bodies, and inhibition of the confluent locule, and these malformed structures may be partially responsible for the decreased anther dehiscence rate and reduced pollen shedding of the anthers in LYPJ. In contrast, the anther wall and pollen development of SY63 were not substantially changed under HT. Conclusions Our results suggest that disturbed anther walls and pollen development are responsible for the reduced spikelet fertility and grain yield of the tested heat susceptible variety, and noninvasive anthers and pollen formation in response to HT were associated with improved heat tolerance.

Comparative analysis of the complete mitochondrial genome sequences and anther development cytology between maintainer and Ogura-type cytoplasm male-sterile cabbage (B. oleracea Var. capitata)

Background Cytoplasmic male sterility (CMS) has been widely used for commercial F1 hybrid seeds production. CMS is primarily caused by chimeric genes in mitochondrial genomes. However, which specific stages of anther development in cabbage are affected by the chimeric genes remain unclear. Results In the present study, the complete mitochondrial genomes were sequenced and assembled for the maintainer and Ogura CMS cabbage lines. The genome size of the maintainer and Ogura CMS cabbage are 219,962 bp and 236,648 bp, respectively. There are 67 and 69 unknown function ORFs identified in the maintainer and Ogura CMS cabbage mitochondrial genomes, respectively. Four orfs, orf102a, orf122b, orf138a and orf154a were specifically identified in the Ogura CMS mitochondrial genome, which were likely generated by recombination with Ogura type radish during breeding process. Among them, ORF138a and ORF154a possessed a transmembrane structure, and orf138a was co-transcribed with the atp8 and trnfM genes. orf154a is partially homologous to the ATP synthase subunit 1 (atpA) gene. Both these genes were likely responsible for the CMS phenotype. In addition, cytological sections showed that the abnormal proliferation of tapetal cells might be the immediate cause of cytoplasmic male-sterility in Ogura CMS cabbage lines. RNA-seq results showed that orf138a and orf154a in Ogura CMS might influence transcript levels of genes in energy metabolic pathways. Conclusions The presence of orf138a and orf154a lead to increased of ATPase activity and ATP content by affecting the transcript levels of genes in energy metabolic pathways, which could provide more energy for the abnormal proliferation of tapetal cells. Our data provides new insights into cytoplasmic male-sterility from whole mitochondrial genomes, cytology of anther development and transcriptome data.

24-nt phasiRNAs move from tapetal to meiotic cells in maize anthers

In maize, 24-nt phased, secondary small interfering RNAs (phasiRNAs) are abundant in meiotic stage anthers, but their distribution and functions are not precisely known. Using laser capture microdissection we analyzed tapetal cells, meiocytes, and other somatic cells at several stages of anther development to establish the timing of 24-PHAS precursor transcripts and the 24-nt phasiRNA products. By integrating RNA and small RNA (sRNA) profiling plus single-molecule and sRNA FISH (smFISH or sRNA-FISH) spatial detection, we demonstrate that the tapetum is the primary site of 24-PHAS precursor and Dcl5 transcripts and the resulting 24-nt phasiRNAs. Interestingly, 24-nt phasiRNAs accumulate in all cell types, with the highest levels in meiocytes, followed by tapetum. Our data support the conclusion that 24-nt phasiRNAs are mobile from tapetum to meiocytes and to other somatic cells. We discuss possible roles for 24-nt phasiRNAs in anther cell types.

Pre-meiotic 21-nucleotide reproductive phasiRNAs emerged in seed plants and diversified in flowering plants

Plant small RNAs are important regulatory elements that fine-tune gene expression and maintain genome integrity by silencing transposons. Reproductive organs of monocots produce abundant phased, small interfering RNAs (phasiRNAs). The 21-nt reproductive phasiRNAs triggered by miR2118 are highly enriched in pre-meiotic anthers, and have been found in multiple eudicot species, in contrast with prior reports of monocot specificity. The 24-nt reproductive phasiRNAs are triggered by miR2275, and are highly enriched during meiosis in many angiosperms. Here, we report the widespread presence of the 21-nt reproductive phasiRNA pathway in eudicots including canonical and non-canonical microRNA (miRNA) triggers of this pathway. In eudicots, these 21-nt phasiRNAs are enriched in pre-meiotic stages, a spatiotemporal distribution consistent with that of monocots and suggesting a role in anther development. Although this pathway is apparently absent in well-studied eudicot families including the Brassicaceae, Solanaceae and Fabaceae, our work in eudicots supports an earlier singular finding in spruce, a gymnosperm, indicating that the pathway of 21-nt reproductive phasiRNAs emerged in seed plants and was lost in some lineages.

Transcriptome Profile Analysis Reveals the Regulation Mechanism of Stamen Abortion in Handeliodendron bodinieri

Handeliodendron bodinieri has unisexual flowers with aborted stamens in female trees, which can be used to study unisexual flower development in tree species. To elucidate the molecular mechanism of stamen abortion underlying sex differentiation, the stage of stamen abortion was determined by semi-thin sections; results showed that stamen abortion occurred in stage 6 during anther development. In addition, differentially expressed transcripts regulating stamen abortion were identified by comparing the transcriptome of female flowers and male flowers with RNA-seq technique. The results showed that 14 genes related to anther development and meiosis such as HbGPAT, HbAMS, HbLAP5, HbLAP3, and HbTES were down-regulated, and HbML5 was up-regulated. Therefore, this information will provide a theoretical foundation for the conservation, breeding, scientific research, and application of Handeliodendron bodinieri.