Abnormal Tapetum Development Research Articles

Sporophytic control of pollen meiotic progression is mediated by tapetum expression of ABORTED MICROSPORES.

Pollen development is dependent on the tapetum, a sporophytic anther cell layer surrounding the microspores that functions in pollen wall formation but is also essential for meiosis-associated development. There is clear evidence of crosstalk and co-regulation between the tapetum and microspores, but how this is achieved is currently not characterized. ABORTED MICROSPORES (AMS), a tapetum transcription factor, is important for pollen wall formation, but also has an undefined role in early pollen development. We conducted a detailed investigation of chromosome behaviour, cytokinesis, radial microtubule array (RMA) organization, and callose formation in the ams mutant. Early meiosis initiates normally in ams, shows delayed progression after the pachytene stage, and then fails during late meiosis, with disorganized RMA, defective cytokinesis, abnormal callose formation, and microspore degeneration, alongside abnormal tapetum development. Here, we show that selected meiosis-associated genes are directly repressed by AMS, and that AMS is essential for late meiosis progression. Our findings indicate that AMS has a dual function in tapetum-meiocyte crosstalk by playing an important regulatory role during late meiosis, in addition to its previously characterized role in pollen wall formation. AMS is critical for RMA organization, callose deposition, and therefore cytokinesis, and is involved in the crosstalk between the gametophyte and sporophytic tissues, which enables synchronous development of tapetum and microspores.

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.

A novel Arabidopsis gene RGAT1 is required for GA-mediated tapetum and pollen development.

The phytohormone gibberellin (GA) is critical for anther development. RGA, a member of the DELLA family of proteins that are central GA signalling repressors, is a key regulator of male fertility in plants. However, the downstream genes in GA-RGA-mediated anther development remain to be characterised. 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 of function 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 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.

Abnormal tapetum development in hermaphrodites of an androdioecious tree, Tapiscia sinensis.

Tapiscia sinensis Oliv. (Tapisciaceae) has been proven to be a functional androdioecious species with both male and hermaphroditic individuals, and the pollen viability of males is far higher than that of hermaphrodites. To better understand the causes of the low pollen viability in hermaphroditic flowers, different stages of anther development were observed. We found that hermaphroditic flowers exhibit abnormal tapetum development, resulting in low pollen viability. To clarify the underlying molecular mechanism of abnormal tapetum development in hermaphrodites, quantitative real-time PCR analyses were performed. The results revealed that the expression levels of an important transcription factor for tapetum development and function, T. sinensis DYSFUNCTIONAL TAPETUM1 (TsDYT1), and its potential downstream regulatory genes T. sinensis DEFECTIVE in TAPETAL DEVELOPMENT and FUNCTION1 (TsTDF1), T. sinensis ABORTED MICROSPORE (TsAMS) and T. sinensis MALE STERILITY 1 (TsMS1) were all significantly downregulated in hermaphrodites compared with males at some key stages of anther development. The amino acid sequence similarity, expression pattern, gene structure and subcellular localization of these genes were analyzed, and the results indicated functional conservation between T. sinensis and homologues in Arabidopsis thaliana. Next, rapid amplification of cDNA end and thermal asymmetric interlaced PCR were employed to clone the full-length cDNA and promoter sequences of these genes, respectively. In addition, results of yeast two-hybrid analysis showed that TsDYT1 can form heterodimers with TsAMS, and yeast one-hybrid analysis demonstrated that TsDYT1 directly binds to the promoter regions of TsTDF1 and TsMS1. TsTDF1 can directly regulate expression of TsAMS, suggesting that a functionally conserved pathway exists between A. thaliana and T. sinensis to regulate tapetum development. In conclusion, the results suggest that abnormal expression of core transcription factors for tapetum development, including TsDYT1, TsTDF1, TsAMS and TsMS1, plays an important role in the abnormal development of the tapetum in T. sinensis hermaphrodites. Furthermore, a hermaphroditic tapetum with abnormal function causes the low pollen viability of hermaphroditic trees. Our results provide new insight into our understanding of the underlying mechanism of why pollen viability is much higher in males than hermaphrodites of the androdioecious tree T. sinensis.

Abnormal tapetum development and energy metabolism associated with sterility in SaNa-1A CMS of Brassica napus L.

Abnormal tapetum degradation and anther development in cytoplasmic male sterility SaNa-1A are the main reasons for the anther abortion. SaNa-1A is a novel cytoplasmic male sterility (CMS) line of Brassica napus derived from somatic hybrids of B. napus-Sinapis alba, and SaNa-1B is the corresponding maintainer line. Ultrastructural comparison between developing anthers of sterile and maintainer lines revealed abnormal subcellular structure of pollen mother cells (PMCs) in the CMS line. The PMC volume and size of nucleus and nucleolus in the CMS line were smaller than those in the maintainer line. The abnormal tapetum cell development and delayed tapetum degradation inhibited microspore development. Finally, anther abortion in the CMS line occurred. Physiological and biochemical analyses of developing anthers and mitochondria revealed that over-accumulation of reactive oxygen species (ROS) in the SaNa-1A and deficiency in antioxidant enzyme system aggravated the oxidization of membrane lipids, resulting in malondialdehyde (MDA) accumulation in anthers. High MDA content in the CMS line was toxic to the cells. ROS accumulation in SaNa-1A also affected anther development. Abnormal structure and function of terminal oxidase, which participates in the electron transport chain of mitochondrial membrane, were observed and affected the activity of cytochrome c oxidase and F1F0-ATPase, which inhibited ATP biosynthesis. Proline deficiency in SaNa-1A also affected anther development. Few hybridization signals of programmed cell death (PCD) in tetrads of SaNa-1A were identified using TdT-mediated dUTP Nick-End Labeling assay. PCD was not obvious in tapetum cells of SaNa-1A until the unicellular stage. These results validated the cytological differences mentioned above, and proved that abnormal tapetum degradation and anther development in SaNa-1A were the main reasons for the anther abortion.

Transcriptional regulation of male-sterility in 7B-1 male-sterile tomato mutant.

The 7B-1 tomato (Solanum lycopersicum L. cv Rutgers) is a male-sterile mutant with enhanced tolerance to abiotic stress, which makes it a potential candidate for hybrid seed breeding and stress engineering. To underline the molecular mechanism regulating the male-sterility in 7B-1, transcriptomic profiles of the 7B-1 male-sterile and wild type (WT) anthers were studied using mRNA sequencing (RNA-Seq). In total, 768 differentially expressed genes (DEGs) were identified, including 132 up-regulated and 636 down-regulated transcripts. Gene ontology (GO) enrichment analysis of DEGs suggested a general impact of the 7B-1 mutation on metabolic processes, such as proteolysis and carbohydrate catabolic process. Sixteen candidates with key roles in regulation of anther development were subjected to further analysis using qRT-PCR and in situ hybridization. Cytological studies showed several defects associated with anther development in the 7B-1 mutant, including unsynchronized anther maturation, dysfunctional meiosis, arrested microspores, defect in callose degradation and abnormal tapetum development. TUNEL assay showed a defect in programmed cell death (PCD) of tapetal cells in 7B-1 anthers. The present study provides insights into the transcriptome of the 7B-1 mutant. We identified several genes with altered expression level in 7B-1 (including beta-1,3 glucanase, GA2oxs, cystatin, cysteine protease, pectinesterase, TA29, and actin) that could potentially regulate anther developmental processes, such as meiosis, tapetum development, and cell-wall formation/degradation.

Glycerol-3-Phosphate Acyltransferase 3 (OsGPAT3) is required for anther development and male fertility in rice.

Lipid molecules are key structural components of plant male reproductive organs, such as the anther and pollen. Although advances have been made in the understanding of acyl lipids in plant reproduction, the metabolic pathways of other lipid compounds, particularly glycerolipids, are not fully understood. Here we report that an endoplasmic reticulum-localized enzyme, Glycerol-3-Phosphate Acyltransferase 3 (OsGPAT3), plays an indispensable role in anther development and pollen formation in rice. OsGPAT3 is preferentially expressed in the tapetum and microspores of the anther. Compared with wild-type plants, the osgpat3 mutant displays smaller, pale yellow anthers with defective anther cuticle, degenerated pollen with defective exine, and abnormal tapetum development and degeneration. Anthers of the osgpat3 mutant have dramatic reductions of all aliphatic lipid contents. The defective cuticle and pollen phenotype coincide well with the down-regulation of sets of genes involved in lipid metabolism and regulation of anther development. Taking these findings together, this work reveals the indispensable role of a monocot-specific glycerol-3-phosphate acyltransferase in male reproduction in rice.

Histone H2B Monoubiquitination Mediated by HISTONE MONOUBIQUITINATION1 and HISTONE MONOUBIQUITINATION2 Is Involved in Anther Development by Regulating Tapetum Degradation-Related Genes in Rice.

Histone H2B monoubiquitination (H2Bub1) is an important regulatory mechanism in eukaryotic gene transcription and is essential for normal plant development. However, the function of H2Bub1 in reproductive development remains elusive. Here, we report rice (Oryza sativa) HISTONE MONOUBIQUITINATION1 (OsHUB1) and OsHUB2, the homologs of Arabidopsis (Arabidopsis thaliana) HUB1 and HUB2 proteins, which function as E3 ligases in H2Bub1, are involved in late anther development in rice. oshub mutants exhibit abnormal tapetum development and aborted pollen in postmeiotic anthers. Knockout of OsHUB1 or OsHUB2 results in the loss of H2Bub1 and a reduction in the levels of dimethylated lysine-4 on histone 3 (H3K4me2). Anther transcriptome analysis revealed that several key tapetum degradation-related genes including OsC4, rice Cysteine Protease1 (OsCP1), and Undeveloped Tapetum1 (UDT1) were down-regulated in the mutants. Further, chromatin immunoprecipitation assays demonstrate that H2Bub1 directly targets OsC4, OsCP1, and UDT1 genes, and enrichment of H2Bub1 and H3K4me2 in the targets is consistent to some degree. Our studies suggest that histone H2B monoubiquitination, mediated by OsHUB1 and OsHUB2, is an important epigenetic modification that in concert with H3K4me2, modulates transcriptional regulation of anther development in rice.

Deficiency in a very-long-chain fatty acid β-ketoacyl-coenzyme a synthase of tomato impairs microgametogenesis and causes floral organ fusion.

Previously, it was shown that β-ketoacyl-coenzyme A synthase ECERIFERUM6 (CER6) is necessary for the biosynthesis of very-long-chain fatty acids with chain lengths beyond C₂₈ in tomato (Solanum lycopersicum) fruits and C₂₆ in Arabidopsis (Arabidopsis thaliana) leaves and the pollen coat. CER6 loss of function in Arabidopsis resulted in conditional male sterility, since pollen coat lipids are responsible for contact-mediated pollen hydration. In tomato, on the contrary, pollen hydration does not rely on pollen coat lipids. Nevertheless, mutation in SlCER6 impairs fertility and floral morphology. Here, the contribution of SlCER6 to the sexual reproduction and flower development of tomato was addressed. Cytological analysis and cross-pollination experiments revealed that the slcer6 mutant has male sterility caused by (1) hampered pollen dispersal and (2) abnormal tapetum development. SlCER6 loss of function provokes a decrease of n- and iso-alkanes with chain lengths of C₂₇ or greater and of anteiso-alkanes with chain lengths of C₂₈ or greater in flower cuticular waxes, but it has no impact on flower cuticle ultrastructure and cutin content. Expression analysis confirmed high transcription levels of SlCER6 in the anther and the petal, preferentially in sites subject to epidermal fusion. Hence, wax deficiency was proposed to be the primary reason for the flower fusion phenomenon in tomato. The SlCER6 substrate specificity was revisited. It might be involved in elongation of not only linear but also branched very-long-chain fatty acids, leading to production of the corresponding alkanes. SlCER6 implements a function in the sexual reproduction of tomato that is different from the one in Arabidopsis: SlCER6 is essential for the regulation of timely tapetum degradation and, consequently, microgametogenesis.

Studies on Abnormal Hypertrophy of Tapetum caused by Auxins in Rice Plants, especially in Relation to that caused by Low Temperature

It is well known phenomenon that abnormal hypertrophy of tapetum takes place in rice plants encountered with an unfavourable low temperature. In a previous paper we have discribed the phenomenon of abnormal hypertrophy of tapetum might be an incidence controlled by the level of endogenous auxin during the anther development. This experiment was carried out with the purpose to prove if exogenously supplied auxins can induce the same abnomral hypertrophy of tapetum as that caused by the low temperature. Naphthaleneacetic acid (NAA) and 2, 4-dichlorophenoxyacetic acid (2, 4-D) were used at concentrations of 0.1, 1.0 and 10.0 ppm. The young spikes at the period of first and second constructions of micrspores were floated on the solution of chemicals for 24 hr at room temperature. Then the anthers were fixed in FAA and prepared for histological observations by means of the usual paraffin method sectioning about 10μ in thickness. Various types of tapetal abnormalities as shown in figs. 2∼17 were observed in the anthers treated with auxins, especially in the plots of the higher concentrations. Thus it became clear that the auxins used obviously induce or promote the same tapetal hypertrophy as that caused by the low temperature. These results seem to support the concept previously reported by the writers that appearance of the tapetal hypertrophy under the low temperature might have a close relationship with the endogenous level of auxin during the anther development. The interaction between auxins and RNA in connection with the abnormal development of tapetum was discussed quoting Dr. Shibuya's experimental result that the tapetal abnormality was induced by application with uridine-monophosphate or triphosphate plus glucose-1-phosphate.