Anther Development Research Articles

Revealing the role of CCoAOMT1: fine-tuning bHLH transcription factors for optimal anther development.

The tapetum, a crucial innermost layer encompassing male reproductive cells within the anther wall, plays a pivotal role in normal pollen development. The transcription factors (TFs) bHLH010/089/091 redundantly facilitate the rapid nuclear accumulation of DYSFUNCTIONAL TAPETUM 1, a gatekeeper TF in the tapetum. Nevertheless, the regulatory mechanisms governing the activity of bHLH010/089/091 remain unknown. In this study, we reveal that caffeoyl coenzyme A O-methyltransferase 1 (CCoAOMT1) is a negative regulator affecting the nuclear localization and function of bHLH010 and bHLH089, probably through their K259 site. Our findings underscore that CCoAOMT1 promotes the nuclear export and degradation of bHLH010 and bHLH089. Intriguingly, elevated CCoAOMT1 expression resulted in defective pollen development, mirroring the phenotype observed in bhlh010 bhlh089 mutants. Moreover, our investigation revealed that the K259A mutation in the bHLH089 protein disrupted its translocation from the nucleus to the cytosol and impeded its degradation induced by CCoAOMT1. Importantly, transgenic plants with the probHLH089::bHLH089K259A construct failed to rescue proper pollen development or gene expression in bhlh010 bhlh089 mutants. Collectively, these findings emphasize the need to maintain balanced TF homeostasis for male fertility. They firmly establish CCoAOMT1 as a pivotal regulator that is instrumental in achieving equilibrium between the induction of the tapetum transcriptional network and ensuring appropriate anther development.

Environmental regulation of male fertility is mediated through Arabidopsis transcription factors bHLH89, 91, and 10.

Formation of functional pollen and successful fertilization rely on the spatial and temporal regulation of anther and pollen development. This process responds to environmental cues to maintain optimal fertility despite climatic changes. Arabidopsis transcription factors basic helix-loop-helix (bHLH) 10, 89, and 91 were previously thought to be functionally redundant in their control of male reproductive development, however here we show that they play distinct roles in the integration of light signals to maintain pollen development under different environmental conditions. Combinations of the double and triple bHLH10,89,91 mutants were analysed under normal (200 μmol m-2 s-1) and low (50 μmol m-2 s-1) light conditions to determine the impact on fertility. Transcriptomic analysis of a new conditionally sterile bhlh89,91 double mutant shows differential regulation of genes related to sexual reproduction, hormone signal transduction, and lipid storage and metabolism under low light. Here we have shown that bHLH89 and bHLH91 play a role in regulating fertility in response to light, suggesting that they function in mitigating environmental variation to ensure fertility is maintained under environmental stress.

Single-Cell Transcriptome Atlas and Regulatory Dynamics in Developing Cotton Anthers.

Plant anthers are composed of different specialized cell types with distinct roles in plant reproduction. High temperature (HT) stress causes male sterility, resulting in crop yield reduction. However, the spatial expression atlas and regulatory dynamics during anther development and in response to HT remain largely unknown. Here, the first single-cell transcriptome atlas and chromatin accessibility survey in cotton anther are established, depicting the specific expression and epigenetic landscape of each type of cell in anthers. The reconstruction of meiotic cells, tapetal cells, and middle layer cell developmental trajectories not only identifies novel expressed genes, but also elucidates the precise degradation period of middle layer and reveals a rapid function transition of tapetal cells during the tetrad stage. By applying HT, heterogeneity in HT response is shown among cells of anthers, with tapetal cells responsible for pollen wall synthesis are most sensitive to HT. Specifically, HT shuts down the chromatin accessibility of genes specifically expressed in the tapetal cells responsible for pollen wall synthesis, such asQUARTET 3 (QRT3) and CYTOCHROME P450 703A2 (CYP703A2), resulting in a silent expression of these genes, ultimately leading to abnormal pollen wall and male sterility. Collectively, this study provides substantial information on anthers and provides clues for heat-tolerant crop creation.

Heat-responsive microRNAs participate in regulating the pollen fertility stability of CMS-D2 restorer line under high-temperature stress

Anther development and pollen fertility of cytoplasmic male sterility (CMS) conditioned by Gossypium harknessii cytoplasm (CMS-D2) restorer lines are susceptible to continuous high-temperature (HT) stress in summer, which seriously hinders the large-scale application of "three-line" hybrids in production. Here, integrated small RNA, transcriptome, degradome, and hormone profiling was performed to explore the roles of microRNAs (miRNAs) in regulating fertility stability in mature pollens of isonuclear alloplasmic near-isogenic restorer lines NH and SH under HT stress at two environments. A total of 211 known and 248 novel miRNAs were identified, of which 159 were differentially expressed miRNAs (DEMs). Additionally, 45 DEMs in 39 miRNA clusters (PmCs) were also identified, and most highly expressed miRNAs were significantly induced in SH under extreme HT, especially four MIR482 and six MIR6300 family miRNAs. PmC28 was located in the fine-mapped interval of the Rf1 gene and contained two DEMs, gra-miR482_L-2R + 2 and gma-miR2118a-3p_R + 1_1ss18TG. Transcriptome sequencing identified 6281 differentially expressed genes, of which heat shock protein (HSP)-related genes, such as HSP70, HSP22, HSP18.5-C, HSP18.2 and HSP17.3-B, presented significantly reduced expression levels in SH under HT stress. Through integrating multi-omics data, we constructed a comprehensive molecular network of miRNA–mRNA–gene-KEGG containing 35 pairs of miRNA/target genes involved in regulating the pollen development in response to HT, among which the mtr-miR167a_R + 1, tcc-miR167c and ghr-miR390a, tcc-miR396c_L-1 and ghr-MIR169b-p3_1ss6AG regulated the pollen fertility by influencing ARF8 responsible for the auxin signal transduction, ascorbate and aldarate metabolism, and the sugar and lipid metabolism and transport pathways, respectively. Further combination with hormone analysis revealed that HT-induced jasmonic acid signaling could activate the expression of downstream auxin synthesis-related genes and cause excessive auxin accumulation, followed by a cascade of auxin signal transduction, ultimately resulting in pollen abortion. The results provide a new understanding of how heat-responsive miRNAs regulate the stability of fertility restoration for CMS-D2 cotton under heat stress.

The effects of heat stress on male reproduction and tillering in Sorghum bicolor

AbstractDue to global climate change, heat stress is increasingly harming the growth, development, overall biomass, and grain yield of numerous crops. Heat stress impairs pollen development and thus reduces seed set in Sorghum bicolor (L.) Moench; however, the effects of heat stress on anther development at specific stages and tiller formation remain incompletely understood. Here we report that exposure to heat stress [42°C/32°C (day/night)] at pollen mother cell (PMC) and booting stages profoundly disrupts tapetum and pollen development, resulting in a significant decrease in grain yield in sorghum. Sorghum plants subjected to 9 days or less of heat stress at the PMC stage exhibited normal pollen viability, but 12 days of heat stress caused almost complete loss of grain yield and the formation of nonviable pollen grains. Similarly, sorghum plants that were heat‐stressed for 3 days at the booting stage produced few seeds. Further analysis revealed that aberrant tapetum and pollen development contributed to the sterility of pollen in these heat‐stressed plants. Notably, in addition to inhibiting plant height, a 12‐day heat stress at the PMC stage promoted the formation of basal tillers, whereas a 3‐day heat stress at the booting stage stimulated the formation of apical tillers, which helped salvage seed yield under heat stress conditions. Moreover, the application of exogenous auxin promoted the formation of apical tillers and leaf numbers per apical tiller. Collectively, our findings suggest that sorghum is susceptible to heat stress during both early and late anther development, and auxin might be involved in governing the formation of apical tillers.

Carbohydrate metabolism and cytology of S-type cytoplasmic male sterility in wheat.

Cytoplasmic male sterility (CMS) is an important tool for hybrid heterosis utilization. However, the underlying mechanisms still need to be discovered. An adequate supply of nutrients is necessary for anther development; pollen abortion would occur if the metabolism of carbohydrates were hampered. In order to better understand the relationship between carbohydrate metabolism disorder and pollen abortion in S-CMS wheat, the submicroscopic structure of wheat anthers was observed using light microscopy and transmission electron microscopy; chloroplast proteome changes were explored by comparative proteomic analysis; sugar measuring and enzyme assays were performed; and the expression patterns of carbohydrate metabolism-related genes were studied using quantitative real-time PCR (qRT-PCR) method. These results indicated that the anther and microspore in S-CMS wheat underwent serious structural damage, including premature tapetum degeneration, nutritional shortage, pollen wall defects, and pollen grain malformations. Furthermore, the number of chloroplasts in the anthers of S-CMS lines decreased significantly, causing abnormal carbohydrate metabolism, and disintegration of osmiophilic granules and thylakoids. Meanwhile, some proteins participating in the Calvin cycle and carbohydrate metabolism were abnormally expressed in the chloroplasts of the S-CMS lines, which might lead to chloroplast dysfunction. Additionally, several key enzymes and genes related to carbohydrate metabolism were significantly inhibited in S-CMS. Based on these results, we proposed a carbohydrate metabolism pathway for anther abortion in S-type cytoplasmic male sterility, which would encourage further exploration of the pollen abortion mechanisms for CMS wheat.

Floral morphology of 'Anand' rapid cycling Brassica oleracea .

Abstract The 'Anand' type of cytoplasmic male sterility (CMS) was introduced to Brassica oleracea through protoplast fusion (Cardi and Earle, 1997), providing a source of male sterility that is of value in producing F1 hybrid varieties of this species without the cold sensitivity of 'Ogura' cytoplasm. In this species, as in others in which it has been introduced, 'Anand' mitochondria are associated with flower abnormalities. The best lines have been used by commercial breeders. This paper describes 'Anand' flower abnormalities in further detail. Rapid cycling brassica with 'Anand' cytoplasm may have promise as a model system for research on floral patterning. Materials and Methods. Three lines of rapid cycling B. oleracea carrying the 'Anand' cytoplasm and one wild type were grown. The flower morphology of 'Anand' lines was classified as good in F25-F-1A, fair in F25-F-6B, and poor in F23-F-1B (Cardi and Earle, 1997). Seedlings were started in an indoor growth room, but were maintained in a naturally lit greenhouse at 20° night, 25° day during flowering. Five flowers on 5 plants of each line were examined. Results. Morphological abnormalities affected all floral parts (Table 1). There was substantial variation among flowers on individual plants and among lines but little within lines. Conclusions. "Good" flowers have large petals that push open the sepals before the carpel elongates. Female fertility could be reduced due to occlusion of the stigma or physical damage caused by the stigma pressing against the end of a sepal and kinking the style. Female fertility in some flowers was unlikely because the carpel suture failed to fuse. In some cases the edge of the split carpel was sepal-like, consistent with the absence of SEPALLATA expression. The stigma on a sepal represents the converse. Stamens were most strongly affected, with outer stamens most reduced. This whorl is the one most frequently suppressed in flowering plants (Masters, 1869). The number of stamens was reduced to varying degrees. First to be lost was one or both of the pair outside the petals. Then the outer pair inside the petals was affected. One or both of these stamens could either be lost or doubled to give 3 to 5 stamens. Again a positional cue for stamens appears to be weakened. Anther development was often limited, the expected and desired consequence of the Anand genotype. Some undehisced " good " anthers contained pollen. There is variation even among the lines initially described as " good ". Further selection among these may help identify those that are most promising for further use in hybrid production.

Cytoplasmic male sterility and abortive seed traits generated through mitochondrial genome editing coupled with allotopic expression of atp1 in tobacco.

Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encoded atp1 gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenous atp1 gene was eliminated (Δatp1) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/Δatp1 plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/Δatp1 plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent Δatp1 events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible for atp1 elimination and genome rearrangement in response to exposure to the atp1-targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment.

Barley TAPETAL DEVELOPMENT and FUNCTION1 (HvTDF1) gene reveals conserved and unique roles in controlling anther tapetum development in dicot and monocot plants.

The anther tapetum helps control microspore release and essential components for pollen wall formation. TAPETAL DEVELOPMENT and FUNCTION1 (TDF1) is an essential R2R3 MYB tapetum transcription factor in Arabidopsis thaliana; however, little is known about pollen development in the temperate monocot barley. Here, we characterize the barley (Hordeum vulgare L.) TDF1 ortholog using reverse genetics and transcriptomics. Spatial/temporal expression analysis indicates HvTDF1 has tapetum-specific expression during anther stage 7/8. Homozygous barley hvtdf1 mutants exhibit male sterility with retarded tapetum development, delayed tapetum endomitosis and cell wall degeneration, resulting in enlarged, vacuolated tapetum surrounding collapsing microspores. Transient protein expression and dual-luciferase assays show TDF1 is a nuclear-localized, transcription activator, that directly activates osmotin proteins. Comparison of hvtdf1 transcriptome data revealed several pathways were delayed, endorsing the observed retarded anther morphology. Arabidopsis tdf1 mutant fertility was recovered by HvTDF1, supporting a conserved role for TDF1 in monocots and dicots. This indicates that tapetum development shares similarity between monocot and dicots; however, barley HvTDF1 appears to uniquely act as a modifier to activate tapetum gene expression pathways, which are subsequently also induced by other factors. Therefore, the absence of HvTDF1 results in delayed developmental progression rather than pathway failure, although inevitably still results in pollen degeneration.

BRI1 EMS SUPPRESSOR1 genes regulate abiotic stress and anther development in wheat (Triticum aestivum L.).

BRI1 EMS SUPPRESSOR1 (BES1) family members are crucial downstream regulators that positively mediate brassinosteroid signaling, playing vital roles in the regulation of plant stress responses and anther development in Arabidopsis. Importantly, the expression profiles of wheat (Triticum aestivum L.) BES1 genes have not been analyzed comprehensively and systematically in response to abiotic stress or during anther development. In this study, we identified 23 BES1-like genes in common wheat, which were unevenly distributed on 17 out of 21 wheat chromosomes. Phylogenetic analysis clustered the BES1 genes into four major clades; moreover, TaBES1-3A2, TaBES1-3B2 and TaBES1-3D2 belonged to the same clade as Arabidopsis BES1/BZR1 HOMOLOG3 (BEH3) and BEH4, which participate in anther development. The expression levels of 23 wheat BES1 genes were assessed using real-time quantitative PCR under various abiotic stress conditions (drought, salt, heat, and cold), and we found that most TaBES1-like genes were downregulated under abiotic stress, particularly during drought stress. We therefore used drought-tolerant and drought-sensitive wheat cultivars to explore TaBES1 expression patterns under drought stress. TaBES1-3B2 and TaBES1-3D2 expression was high in drought-tolerant cultivars but substantially repressed in drought-sensitive cultivars, while TaBES1-6D presented an opposite pattern. Among genes preferentially expressed in anthers, TaBES1-3B2 and TaBES1-3D2 expression was substantially downregulated in thermosensitive genic male-sterile wheat lines compared to common wheat cultivar under sterile conditions, while we detected no obvious differences under fertile conditions. This result suggests that TaBES1-3B2 and TaBES1-3D2 might not only play roles in regulating drought tolerance, but also participate in low temperature-induced male sterility.

Potential candidate genes and pathways related to cytoplasmic male sterility in Dianthus spiculifolius as revealed by transcriptome analysis.

We introduced the candidate gene DsHSP70 into Arabidopsis thaliana, resulting in male gametophyte sterility and abnormal degeneration of sepals and petals. Cytoplasmic male sterility (CMS) is a useful tool for hybrid production. However, the regulatory mechanism of CMS in Dianthus spiculifolius remains unclear. In this study, we investigated whether male-sterile line of D. spiculifolius has a malformed tapetum and fails to produce normal fertile pollen. RNA sequencing technology was used to compare the gene expression patterns of the D. spiculifolius male-sterile line and its male fertility maintainer line during anther development. A total of 12,365 differentially expressed genes (DEGs) were identified, among which 1765 were commonly expressed in the S1, S2 and S3 stages. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that these DEGs were mainly involved in oxidation-reduction processes, signal transduction and programmed cell death. Additionally, weighted correlation network analysis (WGCNA) showed that three modules may be related to male sterility. A putative regulatory pathway for the male sterility traits was constructed based on the reproductive development network. After introducing the candidate DsHSP70 gene into Arabidopsis thaliana, we found that overexpressing plants showed anther abortion and shorter filaments, and accompanied by abnormal degeneration of sepals and petals. In summary, our results identified potential candidate genes and pathways related to CMS in D. spiculifolius, providing new insights for further research on the mechanism of male sterility.

OsTKPR1 proteins with a single amino acid substitution fail the synthesis of a specific sporopollenin precursor and cause abnormal exine and pollen development in rice

Fatty acid derivatives are key components of rice pollen exine. The synthesis of aliphatic sporopollenin precursors are initiated in the plastids of the tapetal cells, followed by multiple-step reactions conducted in the endoplasmic reticulum (ER). However, the relative contribution of different precursors to the precise structure of sporopollenin remains largely elusive, let alone the underlying mechanism. Here, we report that two complete male sterile mutants ostkpr1–3 (Tetraketide α-pyrone reductase 1–3, with OsTKPR1P124S substitution) and ostkpr1–4 (with truncated OsTKPR1stop) are defective in pollen exine, Ubisch body and anther cuticle development where ostkpr1–4 display severer phenotypes. Remarkably, OsTKPR1 could produce reduced hydroxylated tetraketide α-pyrone and reduced tetraketide α-pyrone, whereas OsTKPR1P124S fails to produce the latter. Pairwise interaction assays show that mutated OsTKPR1P124S is able to integrate into a recently characterized metabolon, thus its altered catalytic activity is not due to dis-integrity of the metabolon. In short, we find that reduced tetraketide α-pyrone is a key sporopollenin precursor required for normal exine formation, and the conserved 124th proline of OsTKPR1 is essential for the reduction activity. Therefore, this study provided new insights into the sporopollenin precursor constitution critical for exine formation.

Comparative transcriptome analysis reveals the involvement of an MYB transcriptional activator, SmMYB108, in anther dehiscence in eggplant.

Male sterility is a highly attractive agronomic trait as it effectively prevents self-fertilization and facilitates the production of high-quality hybrid seeds in plants. Timely release of mature pollen following anther dehiscence is essential for stamen development in flowering plants. Although several theories have been proposed regarding this, the specific mechanism of anther development in eggplant remains elusive. In this study, we selected an R2R3-MYB transcription factor gene, SmMYB108, that encodes a protein localized primarily in the nucleus by comparing the transcriptomics of different floral bud developmental stages of the eggplant fertile line, F142. Quantitative reverse transcription polymerase chain reaction revealed that SmMYB108 was preferentially expressed in flowers, and its expression increased significantly on the day of flowering. Overexpression of SmMYB108 in tobacco caused anther dehiscence. In addition, we found that SmMYB108 primarily functions as a transcriptional activator via C-terminal activation (amino acid 262-317). Yeast one-hybrid and dual-luciferase reporter assays revealed that genes (SmMYB21, SmARF6, and SmARF8) related to anther development targeted the SmMYB108 promoter. Overall, our results provide insights into the molecular mechanisms involved in the regulation of anther development by SmMYB108.

Genome-Wide Analysis of the MADS-box Gene Family and Expression Analysis during Anther Development in Salvia miltiorrhiza

MADS-box genes constitute a large family of transcription factors that play important roles in plant growth and development. However, our understanding of MADS-box genes involved in anther development and male sterility in Salvia miltiorrhiza is still limited. In this study, 63 MADS-box genes were identified from the genome of the male sterility ecotype Sichuan S. miltiorrhiza (S. miltiorrhiza_SC) unevenly distributed among eight chromosomes. Phylogenetic analysis classified them into two types and 17 subfamilies. They contained 1 to 12 exons and 10 conserved motifs. Evolution analysis showed that segmental duplication was the main force for the expansion of the SmMADS gene family, and duplication gene pairs were under purifying selection. Cis-acting elements analysis demonstrated that the promoter of SmMADS genes contain numerous elements associated with plant growth and development, plant hormones, and stress response. RNA-seq showed that the expression levels of B-class and C-class SmMADS genes were highly expressed during anther development, with SmMADS11 likely playing an important role in regulating anther development and male fertility in S. miltiorrhiza_SC. Overall, this study provides a comprehensive analysis of the MADS-box gene family in S. miltiorrhiza, shedding light on their potential role in anther development and male sterility.

BOTRYOID POLLEN 1 regulates ROS-triggered PCD and pollen wall development by controlling UDP-sugar homeostasis in rice.

Uridine diphosphate (UDP)-sugars are important metabolites involved in the biosynthesis of polysaccharides and may be important signaling molecules. UDP-glucose 4-epimerase (UGE) catalyzes the interconversion between UDP-Glc and UDP-Gal, whose biological function in rice (Oryza sativa) fertility is poorly understood. Here, we identify and characterize the botryoid pollen 1 (bp1) mutant and show that BP1 encodes a UGE that regulates UDP-sugar homeostasis, thereby controlling the development of rice anthers. The loss of BP1 function led to massive accumulation of UDP-Glc and imbalance of other UDP-sugars. We determined that the higher levels of UDP-Glc and its derivatives in bp1 may induce the expression of NADPH oxidase genes, resulting in a premature accumulation of reactive oxygen species (ROS), thereby advancing programmed cell death (PCD) of anther walls but delaying the end of tapetal degradation. The accumulation of UDP-Glc as metabolites resulted in an abnormal degradation of callose, producing an adhesive microspore. Furthermore, the UDP-sugar metabolism pathway is not only involved in the formation of intine but also in the formation of the initial framework for extine. Our results reveal how UDP-sugars regulate anther development and provide new clues for cellular ROS accumulation and PCD triggered by UDP-Glc as a signaling molecule.

The floral biology and the role of staminal connective appendages during pollination of the endoparasite Bdallophytum americanum (Cytinaceae).

Bdallophytum americanum (Cytinaceae) is an endoparasitic plant species, meaning only the flowers emerge from the host during the reproductive season. Reports on the pollination biology of this species state that its primary pollinators are carrion flies attracted by the smell of the flowers and nectar as a reward. However, the functional role of one of the most outstanding attributes of B. americanum has been neglected. These are the staminal appendages formed by the apical overgrowth of connective tissue during anther development. To determine whether these staminal appendages play a role in pollination, we monitored a nectarless population of B. americanum. We described the inflorescence emergence, floral movements, and pollination and performed field experiments to test whether the absence of the staminal connective appendages affected the visitation frequency. Male inflorescences emerge early, and both male and female flowers open during the day and do not close. Hoverflies are the most frequent visitors to both floral sexes and carry the most pollen. Moreover, the movement of staminal appendages matching the pollen viability changes is reported for the first time. The staminal appendages are the structures where pollinators land before foraging. The field experiments showed that the visitation frequency decreased sharply without staminal appendages. As a landing platform, the staminal connective appendages in B. americanum are crucial for pollinator positioning and collecting viable pollen.

Industrial chicory genome gives insights into the molecular timetable of anther development and male sterility.

Industrial chicory (Cichorium intybus var. sativum) is a biannual crop mostly cultivated for extraction of inulin, a fructose polymer used as a dietary fiber. F1 hybrid breeding is a promising breeding strategy in chicory but relies on stable male sterile lines to prevent self-pollination. Here, we report the assembly and annotation of a new industrial chicory reference genome. Additionally, we performed RNA-Seq on subsequent stages of flower bud development of a fertile line and two cytoplasmic male sterile (CMS) clones. Comparison of fertile and CMS flower bud transcriptomes combined with morphological microscopic analysis of anthers, provided a molecular understanding of anther development and identified key genes in a range of underlying processes, including tapetum development, sink establishment, pollen wall development and anther dehiscence. We also described the role of phytohormones in the regulation of these processes under normal fertile flower bud development. In parallel, we evaluated which processes are disturbed in CMS clones and could contribute to the male sterile phenotype. Taken together, this study provides a state-of-the-art industrial chicory reference genome, an annotated and curated candidate gene set related to anther development and male sterility as well as a detailed molecular timetable of flower bud development in fertile and CMS lines.

Spatial distribution of three ARGONAUTEs regulates the anther phasiRNA pathway

Argonaute protein (AGO) in association with small RNAs is the core machinery of RNA silencing, an essential mechanism for precise development and defense against pathogens in many organisms. Here, we identified two AGOs in rice anthers, AGO1b and AGO1d, that interact with phased small interfering RNAs (phasiRNAs) derived from numerous long non-coding RNAs. Moreover, 3D-immunoimaging and mutant analysis indicated that rice AGO1b and AGO1d cell type-specifically regulate anther development by acting as mobile carriers of these phasiRNAs from the somatic cell layers to the germ cells in anthers. Our study also highlights a new mode of reproductive RNA silencing via the specific nuclear and cytoplasmic localization of three AGOs, AGO1b, AGO1d, and MEL1, in rice pollen mother cells.

Comprehensive identification of polygalacturonases in cotton: Genomic analysis, potential regulatory mechanisms and expression patterns in anthers

Polygalacturonases (PGs) are important plant cell wall degrading enzymes that catalyze pectin hydrolysis and are essential during almost all stages of plant development. However, the functional properties of cotton PGs, especially their expression patterns during reproductive organ development, remain elusive. Herein, the systematic and comprehensive identification and analysis of cotton PGs were conducted. The identified PGs were divided into six groups with conserved gene structures and motifs. The purifying selection was their primary evolutionary force. Meanwhile, cis-elements, TFs, and miRNAs related to stress and tissue-specific development, especially anther development, were identified. Transcriptome and qRT-PCR analysis revealed that many GhPGs were expressed in response to environmental stress. They also exhibited tissue specificity, especially at various stages of anther development. Many genes involved in anther development were co-expressed with GhPGs. This study provides valuable information and novel insights for further exploring the function of cotton PGs and their implementation in cotton improvement.

Identification and Cloning of a Putative Male Fertility Gene Encoding an Oxidosqualene Cyclase in Qingke

Anther development is crucial for controlling crop fertility. To elucidate the underlying mechanisms of reproductive development of highland barley (called Qingke in Chinese), two main Qingke cultivars, Zangqing 2000 and Ximala 22, were utilized. Transcriptome analysis showed that lipid, sugar and phenylpropane metabolisms might be the major pathways associated with Qingke male fertility by analyzing the possible common DEGs before anther maturation in both varieties. Additionally, 26 genes related to crop genic male sterility were screened to identify homologous genes for Qingke male sterile lines. Among them, HORVU.MOREX.r3.7HG0634780 (HvtOSC12), an oxidosqualene cyclase, was highlighted as a candidate gene for affecting Qingke male fertility, as it is highly and especially expressed before Qingke anther maturation. Furthermore, HvtOSC12 (including promoter sequence) was cloned by homology-based cloning. The further bioinformatic analysis deduced that MYBs might be one of the transcription factors affecting expression of HvtOSC12 by responding to environmental changes. These results might lay a foundation for the potential applications for the creation of environment-sensitive genic male sterility in Qingke.