绒毡层发育、花粉发育

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Pollen formation and development during the life cycle of flowering plant are crucial for maintaining reproductive and genetic diversity. In this study, an R2R3MYB family transcription factor, SlTDF1 (SlMYB35), was predominantly expressed in stamens. Repressed expression of SlTDF1 results in a delay in the degradation of the anther tapetum in tomatoes, which in turn leads to the formation of abnormal pollen, including a reduction in the number of single-fruit seeds and fertility when compared to wild-type plants. Analysis of paraffin sections demonstrated that SlTDF1 is a crucial factor in the maturation of tomato pollen. Further analysis of the transcriptomic data revealed that downregulation of the SlTDF1 gene significantly suppressed the expression of genes related to sugar metabolism and anther development. The findings of this study indicated that SlTDF1 plays a pivotal role in regulating tomato pollen development. Moreover, these findings provide a genetic resource for male sterility in tomato plants.

In flowering plants, pollen development is a key process that is essential for sexual reproduction and seed set. Molecular and genetic studies indicate that pollen development is coordinatedly regulated by both gametophytic and sporophytic factors. Tapetum, the somatic cell layer adjacent to the developing male meiocytes, plays an essential role during pollen development. In the early anther development stage, the tapetal cells secrete nutrients, proteins, lipids, and enzymes for microsporocytes and microspore development, while initiating programmed cell death to provide critical materials for pollen wall formation in the late stage. Therefore, disrupting tapetum specification, development, or function usually leads to serious defects in pollen development. In this review, we aim to summarize the current understanding of tapetum-mediated pollen development and illuminate the underlying molecular mechanism in Arabidopsis thaliana.

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In plants, timely degeneration of tapetal cells is essential for providing nutrients and other substances to support pollen development. Rapid alkalinization factors (RALFs) are small, cysteine-rich peptides known to be involved in various aspects of plant development and growth, and defense against biotic and abiotic stresses. However, the functions of most of them remain unknown, while no RALF has been reported to involve in tapetum degeneration. In this study, we demonstrated that a novel cysteine-rich peptide EaF82 isolated from shy-flowering ‘Golden Pothos’ plants is a RALF-like peptide and displays alkalinizing activity. Its heterologous expression in Arabidopsis delayed tapetum degeneration and reduced pollen production and seed yields. RNAseq, RT-qPCR and biochemical analyses showed that overexpressing EaF82 down-regulated a group of genes involved in pH changes, cell wall modifications, tapetum degeneration and pollen maturation as well as seven endogenous Arabidopsis RALF genes, and decreased proteasome activity and ATP levels. Yeast two-hybrid screening identified AKIN10, a subunit of energy-sensing SnRK1 kinase, to be its interacting partner. Our study reveals a possible regulatory role for RALF peptide in tapetum degeneration and suggests that EaF82 action may be mediated through AKIN10 leading to the alteration of transcriptome and energy metabolism, thereby causing ATP deficiency and impairing pollen development.

Abstract AP-1 and AP-2 adaptor protein (AP) complexes mediate clathrin-dependent trafficking at the trans-Golgi network (TGN) and the plasma membrane, respectively. Whereas AP-1 is required for trafficking to plasma membrane and vacuoles, AP-2 mediates endocytosis. These AP complexes consist of four subunits (adaptins): two large subunits (β1 and γ for AP-1 and β2 and α for AP-2), a medium subunit μ, and a small subunit σ. In general, adaptins are unique to each AP complex, with the exception of β subunits that are shared by AP-1 and AP-2 in some invertebrates. Here, we show that the two putative Arabidopsis thaliana AP1/2β adaptins co-assemble with both AP-1 and AP-2 subunits and regulate exocytosis and endocytosis in root cells, consistent with their dual localization at the TGN and plasma membrane. Deletion of both β adaptins is lethal in plants. We identified a critical role of β adaptins in pollen wall formation and reproduction, involving the regulation of membrane trafficking in the tapetum and pollen germination. In tapetal cells, β adaptins localize almost exclusively to the TGN and mediate exocytosis of the plasma membrane transporters such as ATP-binding cassette (ABC)G9 and ABCG16. This study highlights the essential role of AP1/2β adaptins in plants and their specialized roles in specific cell types.

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 sterility (TGMS) wheat line YanZhan 4110S displayed delayed tapetum programmed cell death (PCD) and pollen abortive under the hot temperature stress. Combined with RNA-Sequencing data analysis, TaGAMYB was isolated and involved in fertility conversion, 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.

The phytohormone gibberellin (GA) is critical for anther development. RGA, a member of the DELLA family of proteins that are the central GA signaling repressors, is a key regulator of male fertility in plants. However, the downstream genes in GA-RGA-mediated anther development remains to be characterized. We identified RGA Target 1 (RGAT1), a novel Arabidopsis gene, that functions as an important RGA-regulated target in pollen development. RGAT1 is predominantly expressed in the tapetum and microspores during anther stages 8-11, and can be directly activated by RGA and suppressed by GA in inflorescence apices. Both loss- and gain-of-function of RGAT1 led to abnormal tapetum development, resulting in abortive pollen and short siliques. In RGAT1-knockdown and overexpression lines, pollen abortion occurred at stage 10. Loss of RGAT1 function induced the premature degeneration of tapetal cells with defective ER-derived tapetosomes, while RGAT1 overexpression delayed tapetum degeneration. TUNEL assay confirmed that RGAT1 participates in the timely tapetal programmed cell death. Moreover, reducing RGAT1 expression partially rescued the tapetal developmental defects in GA-deficient ga1-3 mutant. Our findings revealed that RGAT1 is a direct target of RGA and plays an essential role in GA-mediated tapetum and pollen development.

The N-terminal acetylation of proteins is a key modification in eukaryotes. However, knowledge of the biological function of N-terminal acetylation modification of proteins in plants is limited. Naa50 is the catalytic subunit of the N-terminal acetyltransferase NatE complex. We previously demonstrated that the absence of Naa50 leads to sterility in Arabidopsis thaliana. In the present study, the lack of Naa50 resulted in collapsed and sterile pollen in Arabidopsis. Further experiments showed that the mutation in Naa50 accelerated programmed cell death in the tapetum. Expression pattern analysis revealed the specific expression of Naa50 in the tapetum cells of anthers at 9-11 stages during pollen development, when tapetal programmed cell death occurred. Reciprocal cross analyses indicated that male sterility in naa50 is caused by sporophytic effects. mRNA sequencing and quantitative PCR of the closed buds showed that the deletion of Naa50 resulted in the upregulation of the cysteine protease coding gene CEP1 and impaired the expression of several genes involved in pollen wall deposition and pollen mitotic division. The collective data suggest that Naa50 balances the degradation of tapetum cells during anther development and plays an important role in pollen development by affecting several pathways.

In this study, anther wall structure and the embryological features of male gametophyte development in Jurinea kilaea from Asteraceae family are described for the first time. Capitula of different sizes containing young flower buds of J. kilaea was collected from Tekirdağ, Saray - Kastro coast in July 2022 – 2024. Anthers separated according to their sizes under a stereo microscope were passed through arising alcohol series and embedded in Hisstore. Toluidine blue O solution was used to stain the sections. Slides were examined with light microscope and photographed by an Olympus E330 camera. In J. kilaea, anthers are tetrasporangiate. Anther wall consists of the outermost epidermis, the endothecium, the middle layer and the innermost tapetum layer. Tapetum cells appear to have 1 or 2 nuclei. Tapetum is plasmodial type and, tapetum cells begin to degenerate towards the end of the tetrad phase. Microsporogenesis and pollen mitosis are generally regular. Asynchrony is observed during meiosis in young anther loci. Generally, decussate type tetrad was observed. Rarely pentads were also observed. Cytoplasmic channels were observed between microspores at different stages of microsporogenesis. The mature pollen grains of J. kilaea are generally composed of three nuclei and have a normal structure. However, there have been instances where pollen grains exhibit an abnormal structure. Pollen sterility ratio was found to be 12.1%.

Recent phylogenetic studies support the monophyly of the tribe Malveae, with floral morphology providing the main basis for clade delimitation. However, embryological data remain underexplored despite their potential contribution to phylogenetic inference. In this study, we analyzed the embryological development of 12 Neotropical Malveae species to investigate anther ontogeny, microsporogenesis, and microgametogenesis. Floral buds and flowers at different stages of development were collected, and standard methodologies for optical microscopy and confocal laser scanning microscopy were employed. The anther wall pattern corresponds to the dicotyledonous type, with unistratified epidermis, endothecium with ring thickening, an ephemeral middle layer, and a binuclear tapetum of the nonsyncytial invasive type. Microsporogenesis involves simultaneous meiosis, resulting in tetrahedral tetrads. After microspore maturation, microgametogenesis begins, with microspore undergoing asymmetric mitosis, giving rise to vegetative and generative cells. Mature pollen grains are bicellular and contain an abundance of starch. The uniformity in the developmental patterns observed among the analyzed species suggests the presence of shared characteristics that may serve as phylogenetic markers. This strengthens the evidence of a close evolutionary relationship among members of the Malveae tribe and highlights the importance of ontogeny in understanding phylogenetic relationships and delimiting clades in Malvaceae.

Propagation of angiosperms mostly relies on sexual reproduction, in which gametophytic development is a pre-requisite. Male gametophytic development requires both gametophytic and sporophytic factors, most importantly early secretion and late programmed cell death of the tapetum. In addition to transcriptional factors, proteins at endomembrane compartments, such as receptor-like kinases (RLKs) and vacuolar proteases control tapetal function. Cellular machinery regulating their distribution is beginning to be revealed. We report here that ADP-RIBOSYLATION FACTOR-A1s (ArfA1s) are critical for tapetum-controlled pollen development. All six ArfA1s in the Arabidopsis genome are expressed during anther development, among which ArfA1b is specific in tapetum and developing microspores. Although ArfA1b loss-of-function showed no pollen defects likely due to redundancy, interfering ArfA1s by a dominant negative (DN) approach in tapetum resulted in tapetal dysfunction and pollen abortion. We further showed that all six ArfA1s are associated with the Golgi and the trans-Golgi network/early endosome (TGN/EE), suggesting their roles in regulating post-Golgi trafficking to the plasma membrane (PM) or to vacuoles. Indeed, we demonstrated that the expression of ArfA1bDN interfered with the targeting of proteins critical for tapetal development. Results presented demonstrate a key role of ArfA1s in tapetum-controlled pollen development by mediating protein targeting through post-Golgi trafficking routes.

SUMMARY Reduction of crop yield due to iron (Fe) deficiency has always been a concern in agriculture. How Fe insufficiency in floral buds affects pollen development remains unexplored. Here, plants transferred to Fe‐deficient medium at the reproductive stage had reduced floral Fe content and viable pollen and showed a defective pollen outer wall, all restored by supplying floral buds with Fe. A comparison of differentially expressed genes (DEGs) in Fe‐deficient leaves, roots, and anthers suggested that changes in several cellular processes were unique to anthers, including increased lipid degradation. Co‐expression analysis revealed that ABORTED MICROSPORES (AMS), DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION1, and BASIC HELIX‐LOOP‐HELIX 089/091/010 encode key upstream transcription factors of Fe deficiency‐responsive DEGs involved in tapetum function and development, including tapetal ROS homeostasis, programmed cell death, and pollen outer wall formation‐related lipid metabolism. Analysis of RESPIRATORY‐BURST OXIDASE HOMOLOG E (RBOHE) gain‐ and loss‐of‐function under Fe deficiency indicated that RBOHE‐ and Fe‐dependent regulation cooperatively control anther reactive oxygen species levels and pollen development. Since DEGs in Fe‐deficient anthers were not significantly enriched in genes related to mitochondrial function, the changes in mitochondrial status under Fe deficiency, including respiration activity, density, and morphology, were probably because the Fe amount was insufficient to maintain proper mitochondrial protein function in anthers. To sum up, Fe deficiency in anthers may affect Fe‐dependent protein function and impact upstream transcription factors and their downstream genes, resulting in extensively impaired tapetum function and pollen development.

Abstract Pollen development is crucial for the fruit setting process of tomatoes, but the underlying regulatory mechanism remains to be elucidated. Here, we report the isolation of one HD-Zip III family transcription factor, SlHB8, whose expression levels decreased as pollen development progressed. SlHB8 knockout using CRISPR/Cas9 increased pollen activity, subsequently inducing fruit setting, whereas overexpression displayed opposite phenotypes. Overexpression lines under control of the 35 s and p2A11 promoters revealed that SlHB8 reduced pollen activity by affecting early pollen development. Transmission electron microscopy and TUNEL analyses showed that SlHB8 accelerated tapetum degradation, leading to collapsed and infertile pollen without an intine and an abnormal exine. RNA-seq analysis of tomato anthers at the tetrad stage showed that SlHB8 positively regulates SPL/NZZ expression and the tapetum programmed cell death conserved genetic pathway DYT1–TDF1–AMS–MYB80 as well as other genes related to tapetum and pollen wall development. In addition, DNA affinity purification sequencing, electrophoretic mobility shift assay, yeast one-hybrid assay and dual-luciferase assay revealed SlHB8 directly activated the expression of genes related to pollen wall development. The study findings demonstrate that SlHB8 is involved in tapetum development and degradation and plays an important role in anther development.

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Apocynaceae is one of the richest flowering plant families, and the complexity of their reproductive structures and morphological diversity of the flowers has been considered central to understanding its evolutionary success and diversification. However, the diversity of its embryological characters is poorly known, especially in taxa that are early diverging in the family, such as the Aspidospermateae tribe. Moreover, the knowledge gap on early divergent taxa forbids the reconstruction of evolutionary trends in Apocynaceae. Here, we investigated the anther wall and pollen development of five species of Aspidosperma using conventional plant anatomy techniques. We found that the primary conditions for the Aspidosperma species are fibrous endothecium, middle layer, and secretory tapetum having one-layered and uninucleate cells, besides simultaneous cytokinesis. We also found pollen grains 4–6 colporate, alternately with pseudocolporate in all the species, with ridges formed by the thickened inequal deposition of the infratectum layer in the exine that delimits aperture regions. The differentiated deposition of exine confers a distinctive appearance to pollen grains and appears to be associated with the hydration/dehydration process during pollination. Similar deposition patterns have been identified in other genera within the tribe Aspidospermateae, suggesting that it may constitute a synapomorphy for the tribe.

The tapetum in the anther wall is essential for plant fertility, secreting many components essential for pollen development. Development of the tapetum is controlled by multiple transcription factors and signaling pathways. UDT1, TIP2, TDR, and EAT1 constitute a sequential regulatory cascade crucial for tapetal differentiation in rice, but UDT1‐ and TIP2‐dependent regulatory networks, particularly in early anther development, remain largely unknown. Functional analysis of knockout mutants and spatial–temporal expression analysis demonstrated overlapping expression of TIP2 and UDT1 in the middle layer and tapetum and that the tip2 mutation was epistatic to udt1. Moreover, TIP2 and UDT1 were shown to heterodimerize to activate the expression of downstream genes essential for early anther development. We identified two genes activated by TIP2‐UDT1, OsUPEX1 and OsUPEX2, predicted to encode galactosyltransferases, that were preferentially expressed in the tapetum. Analysis of their single mutants demonstrated their functional redundancy, while the double knockout mutant revealed their critical roles in tapetum development and function, likely in enabling tapetal secretion. Overall, this study provides insights into the regulation of rice anther development by TIP2 and UDT1 and identifies downstream targets OsUPEX1 and OsUPEX2 essential for tapetum function and rice male fertility.

Rice cytoplasmic male sterility (CMS) provides an exceptional model for studying genetic interaction within plant nuclei given its inheritable trait of non-functional male gametophyte. Gaining a comprehensive understanding of the genes and pathways associated with the CMS mechanism is imperative for improving the vigor of hybrid rice agronomically, such as its productivity. Here, we observed a significant decrease in the expression of a gene named OsRab7 in the anther of the CMS line (SJA) compared to the maintainer line (SJB). OsRab7 is responsible for vesicle trafficking and loss function of OsRab7 significantly reduced pollen fertility and setting rate relative to the wild type. Meanwhile, over-expression of OsRab7 enhanced pollen fertility in the SJA line while a decrease in its expression in the SJB line led to the reduced pollen fertility. Premature tapetum and abnormal development of microspores were observed in the rab7 mutant. The expression of critical genes involved in tapetum development (OsMYB103, OsPTC1, OsEAT1 and OsAP25) and pollen development (OsMSP1, OsDTM1 and OsC4) decreased significantly in the anther of rab7 mutant. Reduced activities of the pDR5::GUS marker in the young panicle and anther of the rab7 mutant were also observed. Furthermore, the mRNA levels of genes involved in auxin biosynthesis (YUCCAs), auxin transport (PINs), auxin response factors (ARFs), and members of the IAA family (IAAs) were all downregulated in the rab7 mutant, indicating its impact on auxin signaling and distribution. In summary, these findings underscore the importance of OsRab7 in rice pollen development and its potential link to cytoplasmic male sterility.

The development of pollen is critical to male reproduction in flowering plants. Acyl-CoA synthetase (ACOS) genes play conserved functions in regulating pollen development in various plants. Our previous work found that knockout of the SlACOS1 gene in tomato might decrease fruit setting. The current study further revealed that SlACOS1 was important to pollen development and male fertility. The SlACOS1 gene was preferentially expressed in the stamen of the flower with the highest expression at the tetrad stage of anther development. Mutation of the SlACOS1 gene by the CRISPR/Cas9-editing system reduced pollen number and viability as well as fruit setting. The tapetum layer exhibited premature degradation and the pollen showed abnormal development appearing irregular, shriveled, or anucleate in Slacos1 mutants at the tetrad stage. The fatty acid metabolism in anthers was significantly impacted by mutation of the SlACOS1 gene. Furthermore, targeted fatty acids profiling using GC-MS found that contents of most fatty acids except C18:1 and C18:2 were reduced. Yeast complementation assay demonstrated that the substrate preferences of SlACOS1 were C16:0 and C18:0 fatty acids. Male fertility of Slacos1 mutant could be slightly restored by applying exogenous palmitic acid, a type of C16:0 fatty acid. Taken together, SlACOS1 played important roles on pollen development and male fertility by regulating the fatty acid metabolism and the development of tapetum and tetrad. Our findings will facilitate unraveling the mechanism of pollen development and male fertility in tomato.

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Flower development progresses through twelve distinct stages, meticulously regulated to optimize plant reproductive success. At stage 5, the initiation of anther development occurs, which is further categorized into 14 stages divided into two defined phases: phase 1, known as microsporogenesis, and phase 2, termed microgametogenesis—encompassing pollen maturation and anther dehiscence. The maturation of pollen grains must be temporally synchronized with anther dehiscence, with auxin serving as a pivotal spatio-temporal link between these processes, coordinating various aspects of anther development, including stamen elongation, anther dehiscence, and tapetum development. The tapetum, a secretory tissue adjacent to the meiocytes, is essential for nurturing developing pollen grains by secreting components of the pollen wall and ultimately undergoing programmed cell death (PCD). This review primarily focuses on microgametogenesis, the identity and function of the tapetum during the different progression phases, the role of vesicular signaling in delivering external components crucial for pollen grain maturation, and the distinctive process of PCD associated with these developmental processes.

Tapetal cells comprise an anther tissue fundamental to pollen grain development. They are associated with endoreduplication events, which culminate in polyploid and multinucleated cells, high metabolic activity, and different organelle arrangements to support all the development of the pollen grains. Passiflora species present a secretory tapetum, with diversity in the number and size of nuclei. Tapetal cells undergo numerous changes in a short period of development when compared to the plant's life span. To improve our knowledge of tapetum development, tests assessing ploidy levels, anatomy, cytochemistry, transmission electron microscopy, flow cytometry, as well as conventional and molecular cytogenetics were used in Passiflora actinia and P. elegans. The current data show striking differences in nuclear organisation during tapetal cell development, including mono to quadrinucleate cells, and ploidy levels from 2n to 32n. One of the most peculiar features was the atypical behaviour of the endoplasmic reticulum (ER), which accumulated in the cell border, similar to a ‘cER’, as well as large dictyosomes. This endomembrane configuration may be related to the tapetum nutritional network and secretion of compounds at the end of meiosis. Another atypical feature of the ER was the formation of an invagination to establish ‘binucleated’ polyploid cells. This membrane projection appears when the nuclei form two lobes, as well as when it organises a nucleoplasmic reticulum. These data demonstrate that there are important ultrastructural changes in tapetal cells, including organelle arrangements, ploidy levels, and nuclear activity, common to P. actinia and P. elegans, but different from the plant model A. thaliana.

Gametophyte development in angiosperms occurs within diploid sporophytic structures and requires coordinated development; e.g., development of the male gametophyte pollen depends on the surrounding sporophytic tissue, the tapetum. The mechanisms underlying this interaction remain poorly characterized. The peptide CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 19 (CLE19) plays a “braking” role in preventing the harmful overexpression of tapetum transcriptional regulators to ensure normal pollen development in Arabidopsis. However, the CLE19 receptor is unknown. Here, we show that CLE19 interacts directly with the PXY-LIKE1 (PXL1) ectodomain and induces PXL1 phosphorylation. PXL1 is also required for the function of CLE19 in maintaining the tapetal transcriptional regulation of pollen exine genes. Additionally, CLE19 induces the interactions of PXL1 with SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptors required for pollen development. We propose that PXL1 and SERKs act as receptor and coreceptor, respectively, of the extracellular CLE19 signal, thereby regulating tapetum gene expression and pollen development.

Male sterility is classified as either cytoplasmic male sterility (CMS) or genic male sterility (GMS). Generally, CMS involves mitochondrial genomes interacting with the nuclear genome, while GMS is caused by nuclear genes alone. Male sterility is regulated by multilevel mechanisms in which non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and phased small interfering RNAs (phasiRNAs), which have been proven to be critical elements. The development of high-throughput sequencing technology offers new opportunities to evaluate the genetic mechanism of ncRNAs in plant male sterility. In this review, we summarize the critical ncRNAs that regulate gene expression in ways dependent on or independent of hormones, which involve the differentiation of the stamen primordia, degradation of the tapetum, formation of microspores, and the release of pollen. In addition, the key mechanisms of the miRNA–lncRNA–mRNA interaction networks mediating male sterility in plants are elaborated. We present a different perspective on exploring the ncRNA-mediated regulatory pathways that control CMS in plants and create male-sterile lines through hormones or genome editing. A refined understanding of the ncRNA regulatory mechanisms in plant male sterility for the development of new sterile lines would be conducive to improve hybridization breeding.

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Autotetraploid rice is a useful rice germplasm for polyploid rice breeding. However, low fertility limits its commercial production. A neo-tetraploid rice with high fertility was developed from the progenies of crossing between autotetraploid lines by our research group. Our previous study showed that a myeloblastosis (MYB) transcription factor, MOF1, might be associated with the pollen development in tetraploid rice. However, little information is available about its role in pollen development in tetraploid rice. Here, we identified a new haplotype of MOF1 from neo-tetraploid rice and marked it as MOF1a. Transcriptome and qRT-PCR analysis demonstrated that MOF1a highly expressed in anthers, and displayed differential expression in neo-tetraploid rice compared to tetraploid rice line with low pollen fertility. The mutant (mof1a) of MOF1a, which was generated by CRISPR/Cas9 knockout in neo-tetraploid rice, showed low pollen fertility, and also exhibited abnormal tapetum and middle layer development, and defective chromosome behaviors during meiosis. A total of 13 tapetal related genes were found to be up-regulated in meiotic anthers of MOF1a compared with wild type plants by RNA-seq analysis, including CYP703A3, PTC1, and OsABCG26, which had been demonstrated to affect tapetal development. Moreover, 335 meiosis-related genes displayed differential expression patterns at same stage, including nine important meiosis-related genes, such as metallothionein OsMT1a. These results demonstrated that MOF1a plays an important role in pollen development and provides a foundation for understanding the molecular mechanism underlying MOF1a in reproduction of tetraploid rice.

The tomato (Solanum lycopersicum) male sterile 32 (ms32) mutant has been used in hybrid seed breeding programs largely because it produces no pollen and has exserted stigmas. In this study, histological examination of anthers revealed dysfunctional pollen and tapetum development in the ms32 mutant. The ms32 locus was fine mapped to a 28.5 kb interval that encoded four putative genes. Solyc01g081100, a homolog of Arabidopsis bHLH10/89/90 and rice EAT1, was proposed to be the candidate gene of MS32 because it contained a single nucleotide polymorphism (SNP) that led to the formation of a premature stop codon. A codominant derived cleaved amplified polymorphic sequence (dCAPS) marker, MS32D, was developed based on the SNP. Real-time quantitative reverse-transcription PCR showed that most of the genes, which were proposed to be involved in pollen and tapetum development in tomato, were downregulated in the ms32 mutant. These findings may aid in marker-assisted selection of ms32 in hybrid breeding programs and facilitate studies on the regulatory mechanisms of pollen and tapetum development in tomato.

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Photoperiod and temperature-sensitive male sterility rice is an important line for two-line hybrid rice, and the changes in the cultivation temperature strictly control its pollen fertility. However, the mechanism by which temperature variation regulates pollen fertility is still unclear. This study obtained stable fertile PA64S(F) and sterile PA64S(S) rice from PA64S by controlling temperature changes. PA64S(F) shows a normal anther development and fertile pollen under low temperature (21°C), and PA64S(S) shows delayed degradation of the tapetum cells, leading to abnormal pollen wall formation and ubisch development under normal temperature (28°C). The accumulation of reactive oxygen species (ROS) positively correlates with the programmed cell death (PCD) process of tapetum cells. The delayed accumulation of ROS in the PA64S(S) tapetum at early stages leads to a delayed initiation of the PCD process. Importantly, we localized ascorbic acid (ASA) accumulation in the tapetum cells and determined that ASA is a major antioxidant for ROS homeostasis. ROS-inhibited accumulation plants (PA64S-ASA) demonstrated pollen sterility, higher ASA and lower ROS accumulation in the tapetum, and the absence of PCD processes in the tapetum cell. Abnormal changes in the tapetum of PA64S(S) rice disrupted metabolic pathways such as lipid metabolism, cutin and wax synthesis, sugar accumulation, and phenylpropane, affecting pollen wall formation and substance accumulation, suggesting that the timely accumulation of ROS is critical for male fertility. This study highlights the central role of ROS homeostasis in fertility alteration and also provides an avenue to address the effect of environmental temperature changes on pollen fertility in rice.

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Summary Viable pollen is essential for plant reproduction and crop yield. Its production requires coordinated expression at specific stages during anther development, involving early meiosis‐associated events and late pollen wall formation. The ABORTED MICROSPORES (AMS) transcription factor is a master regulator of sporopollenin biosynthesis, secretion and pollen wall formation in Arabidopsis. Here we show that it has complex regulation and additional essential roles earlier in pollen formation. An inducible‐AMS reporter was created for functional rescue, protein expression pattern analysis, and to distinguish between direct and indirect targets. Mathematical modelling was used to create regulatory networks based on wild‐type RNA and protein expression. Dual activity of AMS was defined by biphasic protein expression in anther tapetal cells, with an initial peak around pollen meiosis and then later during pollen wall development. Direct AMS‐regulated targets exhibit temporal regulation, indicating that additional factors are associated with their regulation. We demonstrate that AMS biphasic expression is essential for pollen development, and defines distinct functional activities during early and late pollen development. Mathematical modelling suggests that AMS may competitively form a protein complex with other tapetum‐expressed transcription factors, and that biphasic regulation is due to repression of upstream regulators and promotion of AMS protein degradation.

Sporopollenin is one of the most structurally sophisticated and chemically recalcitrant biopolymers. In higher plants, sporopollenin is the dominant component of exine, the outer wall of pollen grains, and contains covalently linked phenolics that protect the male gametes from harsh environments. Although much has been learned about the biosynthesis of sporopollenin precursors in the tapetum, the nutritive cell layer surrounding developing microspores, little is known about how the biopolymer is assembled on the microspore surface. We identified SCULP1 (SKS clade universal in pollen) as a seed plant conserved clade of the multicopper oxidase family. We showed that SCULP1 in common wheat (Triticum aestivum) is specifically expressed in the microspore when sporopollenin assembly takes place, localized to the developing exine, and binds p-coumaric acid in vitro. Through genetic, biochemical, and 3D reconstruction analyses, we demonstrated that SCULP1 is required for p-coumaroylation of sporopollenin, exine integrity, and pollen viability. Moreover, we found that SCULP1 accumulation is compromised in thermo-sensitive genic male sterile wheat lines and its expression partially restored exine integrity and male fertility. These findings identified a key microspore protein in autonomous sporopollenin polymer assembly, thereby laying the foundation for elucidating and engineering sporopollenin biosynthesis.

Small interfering RNAs (siRNAs) play a crucial role in plant reproduction, yet their mobility and function remain incompletely understood. We report that a large proportion of siRNAs found in pollen of Capsella rubella relies on mobile siRNAs from maternal sporophytic tissues, highlighting the importance of non-cell-autonomous siRNAs in male gametophyte development. Unlike tapetal siRNAs, which guide DNA methylation and require CLASSY3 and DNA-dependent RNA polymerase IV (Pol IV) activity in the tapetum, we found that Pol IV-dependent mobile siRNAs (PMsiRNAs) mainly function post-transcriptionally and do not guide DNA methylation. Nevertheless, PMsiRNAs share key features with tapetal siRNAs, including Pol IV dependency, clustering and a size range of 21–24 nucleotides. Using a grafting approach, we show that sporophytic Pol IV-dependent siRNAs act as non-cell-autonomous mobile signals that trigger PMsiRNA formation through post-transcriptional gene silencing. This process parallels reproductive phased siRNA biogenesis, which is widespread across angiosperms but has been considered absent in Brassicaceae. Loss of PMsiRNAs causes pollen arrest, underscoring their essential role. Together, these findings highlight siRNAs as long-distance communication signals from maternal sporophytic tissues to the male gametophyte with critical functions in developmental regulation. This study shows that many small RNAs in Capsella rubella pollen originate from maternal tissues. These mobile small RNAs support proper pollen development, revealing that non-cell-autonomous small RNAs are crucial for successful plant reproduction.

Plant sexual reproduction involves highly structured and specialized organs: stamens (male) and gynoecium (female, containing ovules). These organs synchronously develop within protective flower buds. Investigating ovules and pollen is crucial for understanding aspects of fertility and sterility in plants. Research on their development and embryogenesis plays a significant role in determining the taxonomic relationships of various species. Paraffin-embedding associated to examination with light microscope showed the development of ovules and pollen grains in Camelina sativa, a key oilseed crop. The findings indicated that the anthers exhibit tetrasporangiate characteristics, with the anther wall consisting of the epidermis, mechanical layer, transitional layer, and tapetum. The microsporogenesis type is simultaneous and microspore tetrads arrange in tetrahedral tetrads. Scanning electron microscope observations showed that mature pollen grains have a tricolporate aperture and are medium-sized, with microreticulate exine ornamentation on the pollen wall. The gynoecium is characterized as bicarpellate, and the ovule in its mature state is classified as amphitropous and bitegmic. The meiosis division of megasporocytes yields a linear tetrad formation. The eight-nucleate embryo sac following the Polygonum type pattern. With a broader systematic perspective, these embryological and palynological features demonstrate evolutionary conservatism within the Brassicaceae, with minor distinctions potentially representing adaptive changes.

Reactive oxygen species (ROS) are signalling molecules that promote programmed cell death in animal and plant systems. However, their role in rice (Oryza sativa L.) anther development is unclear. In this study, we show that lower transcript levels of the metallothionein gene OsMT2b in japonica rice plants obtained by RNA interference (RNAi) resulted in a serious reduction in the seed setting rate. Observations of semi-thin sections of anthers indicated that tapetum degradation initiates early and ends late in OsMT2b-RNAi plants relative to the wild type (WT). Nitroblue tetrazolium staining and measurements of hydrogen peroxide contents showed that ROS contents are higher in OsMT2b-RNAi plants than in WT. Terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assays showed that abnormal programmed cell death in the tapetum results in sterile microspores. In addition, the OsMT2b-RNAi plants were sensitive to photoperiod; they were sterile under natural long-day conditions but almost fully fertile under natural short-day conditions, indicating that OsMT2b integrates photoperiod information into pollen development. The discovery of this rice material may enrich germplasm resources for two-line hybrid rice breeding, and further research may enable its application in two-line hybrid rice breeding.

Tapetal programmed cell death (PCD) is a complex biological process that plays an important role in pollen formation and reproduction. Here, we identified the MYB2 transcription factor expressed in the tapetum from stage 5 to stage 11 that was essential for tapetal PCD and pollen development in Arabidopsis thaliana. Downregulation of MYB2 retarded tapetal degeneration, produced defective pollen, and decreased pollen vitality. EMSA and transcriptional activation analysis revealed that MYB2 acted as an upstream activator and directly regulated expression of the proteases CEP1 and βVPE. The expression of these proteases was lower in the buds of the myb2 mutant. Overexpression of either/both CEP1 or/and βVPE proteases partially recover pollen vitality in the myb2 background. Taken together, our results revealed that MYB2 regulates tapetal PCD and pollen development by directly activating expression of the proteases CEP1 and βVPE. Thus, a transcription factor/proteases regulatory and activated cascade was established for tapetal PCD during another development in Arabidopsis thaliana. Highlight: MYB2 is involved in tapetal PCD and pollen development by directly regulating expression of the protease CEP1 and βVPE and establishes a transcription factor/proteases regulatory and activated cascade.

Summary 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.