Early Anther Development Research Articles

Abnormal Degraded Tapetum1 (ADT1) is required for tapetal cell death and pollen development in rice.

The timely degradation of tapetum, the innermost somatic anther cell layer in flowering plants, is critical for pollen development. Although several genes involved in tapetum development have been characterized, the molecular mechanisms underlying tapetum degeneration remain elusive. Here, we showed that mutation in Abnormal Degraded Tapetum1 (ADT1)resulted in overaccumulation of Reactive Oxygen Species (ROS) and abnormal anther development, causing earlier tapetum Programmed Cell Death (PCD) and pollen abortion. ADT1 encodes a nuclear membrane localized protein, which is strongly expressed in the developing microspores and tapetal cells during early anther development. Moreover, ADT1 could interact with metallothionein MT2b, which was related to ROS scavenging and cell death regulation. These findings indicate that ADT1 is required for proper timing of tapetum PCD by regulating ROS homeostasis, expanding our understanding of the regulatory network of male reproductive development in rice.

OsSRF8 interacts with OsINP1 and OsDAF1 to regulate pollen aperture formation in rice.

In higher plants, mature male gametophytes have distinct apertures. After pollination, pollen grains germinate, and a pollen tube grows from the aperture to deliver sperm cells to the embryo sac, completing fertilization. In rice, the pollen aperture has a single-pore structure with a collar-like annulus and a plug-like operculum. A crucial step in aperture development is the formation of aperture plasma membrane protrusion (APMP) at the distal polar region of the microspore during the late tetrad stage. Previous studies identified OsINP1 and OsDAF1 as essential regulators of APMP and pollen aperture formation in rice, but their precise molecular mechanisms remain unclear. We demonstrate that the Poaceae-specific OsSRF8 gene, encoding a STRUBBELIG-receptor family 8 protein, is essential for pollen aperture formation in Oryza sativa. Mutants lacking functional OsSRF8 exhibit defects in APMP and pollen aperture formation, like loss-of-function OsINP1 mutants. OsSRF8 is specifically expressed during early anther development and initially diffusely distributed in the microsporocytes. At the tetrad stage, OsSRF8 is recruited by OsINP1 to the pre-aperture region through direct protein-protein interaction, promoting APMP formation. The OsSRF8-OsINP1 complex then recruits OsDAF1 to the APMP site to co-regulate annulus formation. Our findings provide insights into the mechanisms controlling pollen aperture formation in cereal species.

The adaptor protein ECAP, the corepressor LEUNIG, and the transcription factor BEH3 interact and regulate microsporocyte generation in Arabidopsis.

Histospecification and morphogenesis of anthers during development in Arabidopsis (Arabidopsis thaliana) are well understood. However, the regulatory mechanism of microsporocyte generation at the pre-meiotic stage remains unclear, especially how archesporial cells are specified and differentiate into 2 cell lineages with distinct developmental fates. SPOROCYTELESS (SPL) is a key reproductive gene that is activated during early anther development and remains active. In this study, we demonstrated that the EAR motif-containing adaptor protein (ECAP) interacts with the Gro/Tup1 family corepressor LEUNIG (LUG) and the BES1/BZR1 HOMOLOG3 (BEH3) transcription factor to form a transcription activator complex, epigenetically regulating SPL transcription. SPL participates in microsporocyte generation by modulating the specification of archesporial cells and the archesporial cell-derived differentiation of somatic and reproductive cell layers. This study illustrates the regulation of SPL expression by the ECAP-LUG-BEH3 complex, which is essential for the generation of microsporocytes. Moreover, our findings identified ECAP as a key transcription regulator that can combine with different partners to regulate gene expression in distinct ways, thereby facilitating diverse processes in various aspects of plant development.

Comprehensive Insight into Tapetum-Mediated Pollen Development in Arabidopsis thaliana.

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.

ZmCCT10-relayed photoperiod sensitivity regulates natural variation in the arithmetical formation of male germinal cells in maize.

Extensive mutational screening studies have documented genes regulating anther and pollen development. Knowledge concerning how formation of male germinal cell is arithmetically controlled in natural populations, under different environmental conditions, is lacking. We counted pollen number within a single anther and a maize-teosinte BC2 S3 recombinant inbred line population to identify ZmCCT10 as a major determinant of pollen number variation. ZmCCT10 was originally identified as a photoperiod-sensitive negative regulator of flowering. ZmCCT10 inactivation, after transposon insertion within its promoter, is proposed to have accelerated maize spread toward higher latitudes, thus allowing temperate maize to flower under long-day conditions. We showed that the active ZmCCT10 allele decreased pollen formation. As different active and inactive ZmCCT10 alleles have been found in natural maize populations, this represents the first report of a gene controlling pollen number in a crop natural population. These findings suggest that higher pollen number, which provides a competitive advantage in open-pollinated populations, may have been one of the major driving forces for the selection of an inactive ZmCCT10 allele during tropical maize domestication. We provide evidence that ZmCCT10 has opposite effects on cell proliferation of archesporial and tapetum cells and it modulates expression of key regulators during early anther development.

Food/Feed and Environmental Risk Assessment of Genetically Modified Glufosinate-tolerant Oilseed Rape MS8, RF3 and MS8 x RF3 for Import, Processing and Feed Uses under Directive 2001/18/EC (Notification C/BE/96/01)

Food/Feed and Environmental Risk Assessment of Genetically Modified Glufosinate-tolerant Oilseed Rape MS8, RF3 and MS8 x RF3 for Import, Processing and Feed Uses under Directive 2001/18/EC (Notification C/BE/96/01)

RMS2 Encoding a GDSL Lipase Mediates Lipid Homeostasis in Anthers to Determine Rice Male Fertility.

Plant male gametogenesis is a coordinated effort involving both reproductive tissues and sporophytic tissues, in which lipid metabolism plays an essential role. Although GDSL esterases/lipases have been well known as key enzymes for many plant developmental processes and stress responses, their functions in reproductive development remain unclear. Here, we report the identification of a rice male sterile2 (rms2) mutant in rice (Oryza sativa), which is completely male sterile due to the defects in tapetum degradation, cuticle formation in sporophytic tissues, and impaired exine and central vacuole development in pollen grains. RMS2 was map-based cloned as an endoplasmic reticulum-localized GDSL lipase gene, which is predominantly transcribed during early anther development. In rms2, a three-nucleotide deletion and one base substitution (TTGT to A) occurred within the GDSL domain, which reduced the lipid hydrolase activity of the resulting protein and led to significant changes in the content of 16 lipid components and numerous other metabolites, as revealed by a comparative metabolic analysis. Furthermore, RMS2 is directly targeted by the male fertility regulators Undeveloped Tapetum1 and Persistent Tapetal Cell1 both in vitro and in vivo, suggesting that RMS2 may serve as a key node in the rice male fertility regulatory network. These findings shed light on the function of GDSLs in reproductive development and provide a promising gene resource for hybrid rice breeding.

The Major Factors Causing the Microspore Abortion of Genic Male Sterile Mutant NWMS1 in Wheat (Triticum aestivum L.).

Male sterility is a valuable trait for genetic research and production application of wheat (Triticum aestivum L.). NWMS1, a novel typical genic male sterility mutant, was obtained from Shengnong 1, mutagenized with ethyl methane sulfonate (EMS). Microstructure and ultrastructure observations of the anthers and microspores indicated that the pollen abortion of NWMS1 started at the early uninucleate microspore stage. Pollen grain collapse, plasmolysis, and absent starch grains were the three typical characteristics of the abnormal microspores. The anther transcriptomes of NWMS1 and its wild type Shengnong 1 were compared at the early anther development stage, pollen mother cell meiotic stage, and binucleate microspore stage. Several biological pathways clearly involved in abnormal anther development were identified, including protein processing in endoplasmic reticulum, starch and sucrose metabolism, lipid metabolism, and plant hormone signal transduction. There were 20 key genes involved in the abnormal anther development, screened out by weighted gene co-expression network analysis (WGCNA), including SKP1B, BIP5, KCS11, ADH3, BGLU6, and TIFY10B. The results indicated that the defect in starch and sucrose metabolism was the most important factor causing male sterility in NWMS1. Based on the experimental data, a primary molecular regulation model of abnormal anther and pollen developments in mutant NWMS1 was established. These results laid a solid foundation for further research on the molecular mechanism of wheat male sterility.

Environmental Risk Assessment of Glufosinate-Tolerant Genetically Modified Oilseed Rape MS8, RF3 and MS8 x RF3 for Import, Processing and Feed Uses under Directive 2001/18/EC (Notification C/BE/96/01)

Environmental Risk Assessment of Glufosinate-Tolerant Genetically Modified Oilseed Rape MS8, RF3 and MS8 x RF3 for Import, Processing and Feed Uses under Directive 2001/18/EC (Notification C/BE/96/01)

Arabidopsis Cys2/His2 zinc-finger protein MAZ1 is essential for intine formation and exine pattern

Cys2/His2 zinc-finger protein (C2H2-ZFP) is widely involved in the reproductive development of plants, but its role in pollen development is still elusive. Here, we identified a pollen-related C2H2-ZFP gene named as MALE FERTILITY-ASSOCIATED ZINC FINGER PROTEIN 1 (MAZ1), which was first isolated from Arabidopsis thaliana. MAZ1 showed a preferential expression pattern in early anther development. Its mutation resulted in aberrant primexine deposition at the tetrad stage, followed by a defective multiple-layer pattern of exine with irregular baculum and no tectum. Furthermore, microspore development was arrested, and no intine layer was formed. These developmental defects led to fertility reduction and pollen abortion. This study reveals the essential role of MAZ1 in pollen wall development.

Transcriptomic and Proteomic Analysis of Shaan2A Cytoplasmic Male Sterility and Its Maintainer Line in Brassica napus.

Cytoplasmic male sterility (CMS) lines are widely used for hybrid production in Brassica napus. The Shaan2A CMS system is one of the most important in China and has been used for decades; however, the male sterility mechanism underlying Shaan2A CMS remains unknown. Here, we performed transcriptomic and proteomic analysis, combined with additional morphological observation, in the Shaan2A CMS. Sporogenous cells, endothecium, middle layer, and tapetum could not be clearly distinguished in Shaan2A anthers. Furthermore, Shaan2A anther chloroplasts contained fewer starch grains than those in Shaan2B (a near-isogenic line of Shaan2A), and the lamella structure of chloroplasts in Shaan2A anther wall cells was obviously aberrant. Transcriptomic analysis revealed differentially expressed genes (DEGs) mainly related to carbon metabolism, lipid and flavonoid metabolism, and the mitochondrial electron transport/ATP synthesis pathway. Proteomic results showed that differentially expressed proteins were mainly associated with carbohydrate metabolism, energy metabolism, and genetic information processing pathways. Importantly, nine gene ontology categories associated with anther and pollen development were enriched among down-regulated DEGs at the young bud (YB) stage, including microsporogenesis, sporopollenin biosynthetic process, and tapetal layer development. Additionally, 464 down-regulated transcription factor (TF) genes were identified at the YB stage, including some related to early anther differentiation such as SPOROCYTELESS (SPL, also named NOZZLE, NZZ), DYSFUNCTIONAL TAPETUM 1 (DYT1), MYB80 (formerly named MYB103), and ABORTED MICROSPORES (AMS). These results suggested that the sterility gene in the Shaan2A mitochondrion might suppress expression of these TF genes in the nucleus, affecting early anther development. Finally, we constructed an interaction network of candidate proteins based on integrative analysis. The present study provides new insights into the molecular mechanism of Shaan2A CMS in B. napus.

Progressive programmed cell death inwards across the anther wall in male sterile flowers of the gynodioecious plant Plantago lanceolata.

A cell death signal is perceived and responded to by epidermal cells first before being conveyed inwards across the anther wall in male sterile Plantago lanceolata flowers. In gynodioecious plants, floral phenotype is determined by an interplay between cytoplasmic male sterility (CMS)-promoting factors and fertility-restoring genes segregating in the nuclear background. Plantago lanceolata exhibits at least four different sterilizing cytoplasms. MS1, a "brown-anther" male sterile phenotype, segregates with a CMSI cytoplasm and a non-restoring nuclear background in P. lanceolata populations. The aim of this study was to investigate the cytology of early anther development in segregating hermaphrodite and male sterile flowers sharing the same CMSI cytoplasm, and to determine if the sterility phenotype correlates with any changes to the normal pattern of programmed cell death (PCD) that occurs during anther development. Cytology shows cellular abnormalities in all four anther wall layers (epidermis, endothecium, middle layer and tapetum), the persistence and enlargement of middle layer and tapetal cells, and the failure of microspore mother cells to complete meiosis in male sterile anthers. In these anthers, apoptotic-PCD occurs earlier than in fertile anthers and is detected in all four cell layers of the anther wall before the middle layer and tapetal cells become enlarged. PCD is separated spatially and temporally within the anther wall, occurring first in epidermal cells before extending radially to cells in the inner anther wall layers. This is the first evidence of a cell death signal being perceived and responded to by epidermal cells first before being conveyed inwards across the anther wall in male sterile plants.

CIK Receptor Kinases Determine Cell Fate Specification during Early Anther Development in Arabidopsis.

Appropriate cell division and differentiation ensure normal anther development in angiosperms. BARELY ANY MERISTEM 1/2 (BAM1/2) and RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), two groups of leucine-rich repeat receptor-like protein kinases, are required for early anther cell specification. However, little is known about the molecular mechanisms underlying these two RLK-mediated signaling pathways. Here, we show that CLAVATA3 INSENSITIVE RECEPTOR KINASEs (CIKs), a group of novel coreceptor protein kinase-controlling stem cell homeostasis, play essential roles in BAM1/2- and RPK2-regulated early anther development in Arabidopsis thaliana The archesporial cells of cik1/2/3 triple and cik1/2/3/4 quadruple mutant anthers perform anticlinal division instead of periclinal division. Defective cell division and specification of the primary and inner secondary parietal cells occur in these mutant anthers. The disordered divisions and specifications of anther wall cells finally result in excess microsporocytes and a lack of one to three parietal cell layers in mutant anthers, resembling rpk2 or bam1/2 mutant anthers. Genetic and biochemical analyses indicate that CIKs function as coreceptors of BAM1/2 and RPK2 to regulate archesporial cell division and determine the specification of anther parietal cells.

Promoters of Arabidopsis Casein kinase I-like 2 and 7 confer specific high-temperature response in anther.

KEY MESSAGE: (1) We systematically analyze the promoter activities of AtCKLs in various tissues; (2) AtCKL2 and AtCKL7 were expressed in early developmental anthers under high temperature (HT) conditions; (3) AtMYB24 may function as a positive regulator of AtCKL2 and AtCKL7 expression under HT. High temperature (HT) can seriously impede plant growth and development, causing severe loss of crop production. In Arabidopsis, AtCKL genes show high similarity to GhCKI, a gene reported to disrupt tapetal programmed cell death in cotton. However, most of AtCKL genes are not well characterized. Here, we systematically analyzed the expression patterns of AtCKLs in various tissues. The expression of AtCKL2 and AtCKL7 was induced in early anther development under HT, which is similar to the case of GhCKI. In silico analysis of AtCKL2 and AtCKL7 promoters indicated that four types of transcription factors (TFs) (MADS, NAC, WRKY and R2R3-MYB) might bind to AtCKL2 and AtCKL7 promoters. Furthermore, three MADS, three NAC, one WRKY, and three R2R3-MYB TFs were up-regulated in stage 1-8 anthers and three R2R3-MYB TFs were up-regulated in stage 9-10 anthers under HT, implying the important roles of R2R3-MYB genes in the response of anthers to HT. Among the R2R3-MYB genes, AtMYB24 showed the similar expression as AtCKL2 and AtCKL7 in the anthers under HT. Additionally, yeast one-hybrid and dual-luciferase reporter system assays verified that AtMYB24 could bind to AtCKL2 and AtCKL7 promoters and activate the expression of these two genes. In brief, this study provides the overall expression profiles of AtCKLs, useful information for unraveling the molecular mechanism of AtCKL2 and AtCKL7 gene expression in early anther development under HT, and important clues for elucidating the mechanism of transcriptional regulation of CKI genes in plant anther under HT, which are critical to the reduction of crop yield loss resulting from HT.

A Rice Glutamyl-tRNA Synthetase Modulates Early Anther Cell Division and Patterning.

Aminoacyl-tRNA synthetases (aaRSs) have housekeeping roles in protein synthesis, but little is known about how these aaRSs are involved in organ development. Here, we report that a rice (Oryza sativa) glutamyl-tRNA synthetase (OsERS1) maintains proper somatic cell organization and limits the overproliferation of male germ cells during early anther development. The expression of OsERS1 is specifically detectable in meristematic layer 2-derived cells of the early anther, and osers1 anthers exhibit overproliferation and disorganization of layer 2-derived cells, producing fused lobes and extra germ cells in early anthers. The conserved biochemical function of OsERS1 in ligating glutamate to tRNAGlu is enhanced by its cofactor aaRS OsARC. Furthermore, metabolomics profiling revealed that OsERS1 is an important node for multiple metabolic pathways, indicated by the accumulation of amino acids and tricarboxylic acid cycle components in osers1 anthers. Notably, the anther defects of the osers1 mutant are causally associated with the abnormal accumulation of hydrogen peroxide, which can reconstitute the osers1 phenotype when applied to wild-type anthers. Collectively, these findings demonstrate how aaRSs affect male organ development in plants, likely through protein synthesis, metabolic homeostasis, and redox status.

The role of receptor-like kinases in regulating plant male reproduction.

RLKs in anther development. The cell-to-cell communication is essential for specifying different cell types during plant growth, development and adaption to the ever-changing environment. Plant male reproduction, in particular, requires the exquisitely synchronized development of different cell layers within the male tissue, the anther. Receptor-like kinases (RLKs) belong to a large group of kinases localized on the cell surfaces, perceiving extracellular signals and thereafter regulating intracellular processes. Here we update the role of RLKs in early anther development by defining the cell fate and anther patterning, responding to the changing environment and controlling anther carbohydrate metabolism. We provide speculation of the poorly characterized ligands and substrates of these RLKs. The conserved and diversified aspects underlying the function of RLKs in anther development are discussed.

Carbonic Anhydrases Function in Anther Cell Differentiation Downstream of the Receptor-Like Kinase EMS1.

Plants extensively employ leucine-rich repeat receptor-like kinases (LRR-RLKs), the largest family of RLKs, to control a wide range of growth and developmental processes as well as defense responses. To date, only a few direct downstream effectors for LRR-RLKs have been identified. We previously showed that the LRR-RLK EMS1 (EXCESS MICROSPOROCYTES1) and its ligand TPD1 (TAPETUM DETERMINANT1) are required for the differentiation of somatic tapetal cells and reproductive microsporocytes during early anther development in Arabidopsis thaliana Here, we report the identification of β-carbonic anhydrases (βCAs) as the direct downstream targets of EMS1. EMS1 biochemically interacts with βCA proteins. Loss of function of βCA genes caused defective tapetal cell differentiation, while overexpression of βCA1 led to the formation of extra tapetal cells. EMS1 phosphorylates βCA1 at four sites, resulting in increased βCA1 activity. Furthermore, phosphorylation-blocking mutations impaired the function of βCA1 in tapetal cell differentiation; however, a phosphorylation mimic mutation promoted the formation of tapetal cells. βCAs are also involved in pH regulation in tapetal cells. Our findings highlight the role of βCA in controlling cell differentiation and provide insights into the posttranslational modification of carbonic anhydrases via receptor-like kinase-mediated phosphorylation.

Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination.

Cell signaling pathways mediated by leucine-rich repeat receptor-like kinases (LRR-RLKs) are essential for plant growth, development, and defense. The EMS1 (EXCESS MICROSPOROCYTES1) LRR-RLK and its small protein ligand TPD1 (TAPETUM DETERMINANT1) play a fundamental role in somatic and reproductive cell differentiation during early anther development in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether other cell surface molecules serve as coregulators of EMS1. Here, we show that SERK1 (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1) and SERK2 LRR-RLKs act redundantly as coregulatory and physical partners of EMS1. The SERK1/2 genes function in the same genetic pathway as EMS1 in anther development. Bimolecular fluorescence complementation, Förster resonance energy transfer, and coimmunoprecipitation approaches revealed that SERK1 interacted biochemically with EMS1. Transphosphorylation of EMS1 by SERK1 enhances EMS1 kinase activity. Among 12 in vitro autophosphorylation and transphosphorylation sites identified by tandem mass spectrometry, seven of them were found to be critical for EMS1 autophosphorylation activity. Furthermore, complementation test results suggest that phosphorylation of EMS1 is required for its function in anther development. Collectively, these data provide genetic and biochemical evidence of the interaction and phosphorylation between SERK1/2 and EMS1 in anther development.

MYB transcription factor gene involved in sex determination in Asparagus officinalis.

Dioecy is a plant mating system in which individuals of a species are either male or female. Although many flowering plants evolved independently from hermaphroditism to dioecy, the molecular mechanism underlying this transition remains largely unknown. Sex determination in the dioecious plant Asparagus officinalis is controlled by X and Y chromosomes; the male and female karyotypes are XY and XX, respectively. Transcriptome analysis of A.officinalis buds showed that a MYB-like gene, Male Specific Expression 1 (MSE1), is specifically expressed in males. MSE1 exhibits tight linkage with the Y chromosome, specific expression in early anther development and loss of function on the X chromosome. Knockout of the MSE1 orthologue in Arabidopsis induces male sterility. Thus, MSE1 acts in sex determination in A.officinalis.

A Rice Ca2+ Binding Protein Is Required for Tapetum Function and Pollen Formation.

In flowering plants, successful male reproduction requires the sophisticated interaction between somatic anther wall layers and reproductive cells. Timely degradation of the innermost tissue of the anther wall layer, the tapetal layer, is critical for pollen development. Ca2+ is a well-known stimulus for plant development, but whether it plays a role in affecting male reproduction remains elusive. Here we report a role of Defective in Exine Formation 1 (OsDEX1) in rice (Oryza sativa), a Ca2+ binding protein, in regulating rice tapetal cell degradation and pollen formation. In osdex1 anthers, tapetal cell degeneration is delayed and degradation of the callose wall surrounding the microspores is compromised, leading to aborted pollen formation and complete male sterility. OsDEX1 is expressed in tapetal cells and microspores during early anther development. Recombinant OsDEX1 is able to bind Ca2+ and regulate Ca2+ homeostasis in vitro, and osdex1 exhibited disturbed Ca2+ homeostasis in tapetal cells. Phylogenetic analysis suggested that OsDEX1 may have a conserved function in binding Ca2+ in flowering plants, and genetic complementation of pollen wall defects of an Arabidopsis (Arabidopsis thaliana) dex1 mutant confirmed its evolutionary conservation in pollen development. Collectively, these findings suggest that OsDEX1 plays a fundamental role in the development of tapetal cells and pollen formation, possibly via modulating the Ca2+ homeostasis during pollen development.