The Post-meiotic Deficicent Anther1 (PDA1) gene is required for post-meiotic anther development in rice

To understand the molecular mechanism of male reproductive development in the model crop rice,we isolated a complete male sterile mutant post-meiotic deficient anther1 (pda1) from a γ-ray-treated rice mutant library.Genetic analysis revealed that the pda1 mutant was controlled by a recessive nucleus gene.The pda1 mutant anther seemed smaller with white appearance.Histological analysis demonstrated that the pda1 mutant anther undergoes normal early tapetum development without obvious altered meiosis.However,...     

The Post-meiotic Deficicent Anther1 （PDA1） gene is required for post-meiotic anther development in rice

To understand the molecular mechanism of male reproductive development in the model crop rice,we isolated a complete male sterile mutant post-meiotic deficient anther1 (pda1) from a γ-ray-treated rice mutant library.Genetic analysis revealed that the pda1 mutant was controlled by a recessive nucleus gene.The pda1 mutant anther seemed smaller with white appearance.Histological analysis demonstrated that the pda1 mutant anther undergoes normal early tapetum development without obvious altered meiosis.However,the pda1 mutant displayed obvious defects in postmeiotic tapetal development,abnormal degeneration occurred in the tapetal cells at stage 9 of anther development.Also we observed abnormal lipidic Ubisch bodies from the tapetal layer of the pda1 mutant,causing no obvious pollen exine formation.RT-PCR analysis indicated that the expression of genes involved in anther development including GAMYB,OsC4 and Wax-deficient anther1 (WDA1) was greatly reduced in the pda1 mutant anther.Using map-based cloning approach,the PDA1 gene was finely mapped between two markers HLF610 and HLF627 on chromosome 6 using 3,883 individuals of F2 population.The physical distance between HLF610 and HLF627 was about 194 kb.This work suggests that PDA1 is required for post-meiotic tapetal development and pollen/microspore formation in rice.     

The RADICLELESS1 gene is required for vascular pattern formation in rice

The molecular mechanisms through which the complex patterns of plant vascular tissues are established are largely unknown. The highly ordered, yet simple, striate array of veins of rice leaves represents an attractive system to study the dynamics underlying pattern formation. Here we show that mutation in the RADICLELESS1 (RAL1) gene results in distinctive vascular pattern defects. In ral1 embryonic scutella, secondary veins are absent and in the prematurely aborted and discontinuous primary veins, cells are misaligned to each other. In ral1 leaves, longitudinal and commissural (transverse) veins display altered spacing and the commissural veins additionally show atypical branching and interruptions in their continuity. The vascular pattern alterations of ral1 occur in the context of normally shaped leaf primordia. Anatomical inspection and analysis of the expression of the procambium specification marker Oshox1-GUS and of the auxin-inducible reporter DR5-GUS demonstrates that all the vascular patterning aberrations of ral1 originate from defects in the procambium, which represents the earliest identifiable stage of vascular development. Furthermore, the ral1 mutant is unique in that procambium formation in leaf primordium development is delayed. Finally, the ral1 vascular patterning distortions are associated with a defective response to auxin and with an enhanced sensitivity to cytokinin. ral1 is the first mutant impaired in both procambium development and vascular patterning to be isolated in a monocot species.     

The rice RAD51C gene is required for the meiosis of both female and male gametocytes and the DNA repair of somatic cells

The RecA/RAD51 family of rice (Oryza sativa) consists of at least 13 members. However, the functions of most of these members are unknown. Here the functional characterization of one member of this family, RAD51C, is reported. Knockout (KO) of RAD51C resulted in both female and male sterility in rice. Transferring RAD51C to the RAD51C-KO line restored fertility. Cytological analyses showed that the sterility of RAD51C-KO plants was associated with abnormal early meiotic processes in both megasporocytes and pollen mother cells (PMCs). PMCs had an absence of normal pachytene chromosomes and had abnormal chromosome fragments. The RAD51C-KO line showed no obvious difference from wild-type plants in mitosis in the anther wall cells, which was consistent with the observation that the RAD51C-KO line did not have obviously abnormal morphology during vegetative development. However, the RAD51C-KO line was sensitive to different DNA-damaging agents. These results suggest that RAD51C is essential for reproductive development by regulating meiosis as well as for DNA damage repair in somatic cells.     

ABNORMAL POLLEN VACUOLATION1 (APV1) is required for male fertility by contributing to anther cuticle and pollen exine formation in maize

Anther cuticle and pollen exine are the major protective barriers against various stresses. The proper functioning of genes expressed in the tapetum is vital for the development of pollen exine and anther cuticle. In this study, we report a tapetum‐specific gene, Abnormal Pollen Vacuolation1 (APV1), in maize that affects anther cuticle and pollen exine formation. The apv1 mutant was completely male sterile. Its microspores were swollen, less vacuolated, with a flat and empty anther locule. In the mutant, the anther epidermal surface was smooth, shiny, and plate‐shaped compared with the three‐dimensional crowded ridges and randomly formed wax crystals on the epidermal surface of the wild‐type. The wild‐type mature pollen had elaborate exine patterning, whereas the apv1 pollen surface was smooth. Only a few unevenly distributed Ubisch bodies were formed on the apv1 mutant, leading to a more apparent inner surface. A significant reduction in the cutin monomers was observed in the mutant. APV1 encodes a member of the P450 subfamily, CYP703A2‐Zm, which contains 530 amino acids. APV1 appeared to be widely expressed in the tapetum at the vacuolation stage, and its protein signal co‐localized with the endoplasmic reticulum (ER) signal. RNA‐Seq data revealed that most of the genes in the fatty acid metabolism pathway were differentially expressed in the apv1 mutant. Altogether, we suggest that APV1 functions in the fatty acid hydroxylation pathway which is involved in forming sporopollenin precursors and cutin monomers that are essential for the development of pollen exine and anther cuticle in maize.     

The Caenorhabditis elegans ADAMTS family gene adt-1 is necessary for morphogenesis of the male copulatory organs.

Remodeling of the extracellular matrix (ECM) is pivotal for various biological processes, including organ morphology and development. The Caenorhabditis elegans male tail has male-specific copulatory organs, the rays and the fan. Ray morphogenesis, which involves a rapid remodeling of the ECM, is an important model of morphogenesis, although its mechanism is poorly understood. ADAMTS (a disintegrin-like and metalloproteinase with thrombospondin type I motifs) is a novel metalloproteinase family that is thought to be an important regulator for ECM remodeling during development and pathological states. We report here that a new C. elegans ADAMTS family gene, adt-1, plays an important regulatory role in ray morphogenesis. Inactivation of the adt-1 gene resulted in morphological changes in the rays as well as the appearance of abnormal protuberances around the rays. In addition, mating ability was remarkably impaired in adt-1 deletion mutant males. Furthermore, we found that the green fluorescent protein reporter driven by the adt-1 promoter was specifically expressed throughout the rays in the male tail. We hypothesize that ADT-1 controls the ray extension process via remodeling of the ECM in the cuticle.     

Chromosome attachment to the spindle in crane-fly spermatocytes requires actin and is necessary to initiate the anaphase-onset checkpoint

Summary We investigated the possible involvement of actin in the attachment of chromosomes to spindles in crane-fly primary spermatocytes. In a previous study, cytochalasin D, an inhibitor of actin polymerisation, prevented bivalent attachment to microtubules when applied at prophase, but did not cause the detachment of already attached bivalents. We were able to detach the already attached bivalents by first treating prometaphase cells with an antitubulin drug, nocodazole, to disrupt spindle microtubules. 2 min after nocodazole addition, we added cytochalasin D, to disrupt actin filaments; then 2 min later nocodazole was removed, and the cells were kept in cytochalasin D until the time of normal anaphase. Double treatment with nocodazole and cytochalasin D blocked reattachment of bivalents to the spindle. Single treatment with nocodazole alone caused chromosome detachment but did not prevent reattachment when nocodazole was washed out. Extended treatment with cytochalasin D alone starting in prometaphase did not cause bivalents to detach from the spindle. These data suggest that actin is needed for attachment of bivalents to spindle microtubules. This protocol is relevant to the anaphase-onset checkpoint. From previous experiments it was argued that the anaphase-onset checkpoint recognises unattached chromosomes only after those chromosomes first interact with (become attached to) the spindle. Our experiments showed that anaphase disjunction occurred at normal times when bivalents were prevented from attaching to the spindle (by adding cytochalasin D in prophase), while anaphase disjunction was greatly delayed when previously attached bivalents were detached (with nocodazole) and then prevented from re-attaching (with cytochalasin D) in the double treated cells. Thus the anaphaseonset checkpoint recognises only those unattached bivalents that previously were attached to the spindle. Other results provided further indication that actin-microtubule interactions are important in spindle organisation. Nocodazole treatment for 4 min caused most microtubules to disappear: bivalents aggregated around remnant microtubules. When cytochalasin D treatment followed nocodazole treatment, remnant spindle microtubules were not seen, suggesting that actin interactions help stabilise those microtubules.Abbreviations CD cytochalasin D - NMBD nuclear-membrane breakdown - NOC nocodazole     

PSA-NCAM in the posterodorsal medial amygdala is necessary for the pubertal emergence of attraction to female odors in male hamsters

During puberty, attention turns away from same-sex socialization to focus on the opposite sex. How the brain mediates this change in perception and motivation is unknown. Polysialylated neural cell adhesion molecule (PSA-NCAM) virtually disappears from most of the central nervous system after embryogenesis, but it remains elevated in discrete regions of the adult brain. One such brain area is the posterodorsal subnucleus of the medial amygdala (MePD). The MePD has been implicated in male sexual attraction, measured here as the preference to investigate female odors. We hypothesize that PSA-NCAM gates hormone-dependent plasticity necessary for the emergence of males' attraction to females. To evaluate this idea, we first measured PSA-NCAM levels across puberty in several brain regions, and identified when female odor preference normally emerges in male Syrian hamsters. We found that MePD PSA-NCAM staining peaks shortly before the surge of pubertal androgen and the emergence of preference. To test the necessity of PSA-NCAM for female odor preference, we infused endo-neuraminidase-N into the MePD to deplete it of PSAs before female odor preference normally appears. This blocked female odor preference, which suggests that PSA-NCAM facilitates behaviorally relevant, hormone-driven plasticity.     

The Fc receptor gamma-chain is necessary and sufficient to initiate signalling through glycoprotein VI in transfected cells by the snake C-type lectin, convulxin.

There is extensive evidence that FcR gamma-chain couples to the collagen receptor glycoprotein VI (GPVI) and becomes phosphorylated on tyrosines upon receptor cross-linking. However, it is not established whether this receptor complex is sufficient to initiate the signalling cascade. We transfected GPVI and the FcR gamma-chain into the human erythroleukaemia cell line K562, which lacks detectable expression of GPVI and the FcR gamma-chain. The results show that GPVI is unable to signal when expressed alone, despite its surface expression, upon stimulation with the snake C-type lectin, convulxin. Coexpression of the FcR gamma-chain confers signalling properties on the receptor. Furthermore, cotransfection of the FcR gamma-chain and two mutant versions of GPVI shows that the transmembrane arginine and cytoplasmic tail of GPVI are necessary for association with the FcR gamma-chain. These results demonstrate that reconstitution of the GPVI-FcR gamma-chain complex in cells expressing the necessary signalling network is sufficient to initiate signalling events in response to convulxin and collagen-related peptide.     

A germ cell specific gene of the ARGONAUTE family is essential for the progression of premeiotic mitosis and meiosis during sporogenesis in rice

The rice (Oryza sativa) genome contains 18 copies of genes of the ARGONAUTE (AGO) family. Although AGO members play important roles in RNA-mediated silencing during plant development, a family member that is specifically involved in sexual reproduction has not been identified in plants. We identified the rice AGO gene MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1) from the analysis of seed-sterile mutants. In the mel1 mutant, chromosome condensation was arrested at early meiotic stages and irregularly sized, multinucleated, and vacuolated pollen mother cells (PMCs) frequently appeared in developing anthers. In addition, histone H3 lysine-9 dimethylation of pericentromeres was rarely reduced and modification of the nucleolar-organizing region was altered in mel1 mutant PMCs. The mutation also affected female germ cell development. These results indicate that the germ cell-specific rice MEL1 gene regulates the cell division of premeiotic germ cells, the proper modification of meiotic chromosomes, and the faithful progression of meiosis, probably via small RNA-mediated gene silencing, but not the initiation and establishment of germ cells themselves.     

The alpha-N-acetyl-glucosaminidase gene is transcriptionally activated in male and female gametes prior to fertilization and is essential for seed development in Arabidopsis

Sugar residues in proteoglycan complexes carry important signalling and regulatory functions in biology. In humans, heparan sulphate is an example of such a complex polymer containing glucosamine and N-acetyl-glucosamine residues and is present in the extracellular matrix. Although heparan sulphate has not been found in plants, the At5g13690 gene encoding the alpha-N-acetyl-glucosaminidase (NAGLU), an enzyme involved in its catabolism, is present in the Arabidopsis genome. Among our collection of embryo-defective lines, a plant was identified in which the T-DNA had inserted into the AtNAGLU gene. The phenotype of atnaglu is an early arrest of seed development without apparent male or female gametophytic effects. These data demonstrated the essential function in Arabidopsis consistent with the contribution of NAGLU to the Sanfilippo syndrome in human. Expression of AtNAGLU in plants was shown to be prevalent during reproductive development. The presence of AtNAGLU mRNA was observed during early and late male gametogenesis and in each cell of the embryo sac at the time of fertilization. After fertilization, AtNAGLU was expressed in the embryo, suspensor, and endosperm until the cotyledonary stage embryo. This precise pattern of expression identifies the cells and tissues where a remodelling of the N-acetyl-glucosamine residues of proteoglycan complexes is occurring. This work provides original evidence of the important role of N-acetyl-glucosamines in plant reproductive development.     

OsTDL1A binds to the LRR domain of rice receptor kinase MSP1, and is required to limit sporocyte numbers

Hybrids lose heterotic yield advantage when multiplied sexually via meiosis. A potential alternative breeding system for hybrids is apospory, where female gametes develop without meiosis. Common among grasses, apospory begins in the nucellus, where aposporous initials (AIs) appear near the sexual megaspore mother cell (MeMC). The cellular origin of AIs is obscure, but one possibility, suggested by the mac1 and msp1 mutants of maize and rice, is that AIs are apomeiotic derivatives of the additional MeMCs that appear when genetic control over sporocyte numbers is relaxed. MULTIPLE SPOROCYTES1 (MSP1) encodes a leucine-rich-repeat receptor kinase, which is orthologous to EXS/EMS1 in Arabidopsis. Like mac1 and msp1, exs/ems1 mutants produce extra sporocytes in the anther instead of a tapetum, causing male sterility. This phenotype is copied in mutants of TAPETUM DETERMINANT1 (TPD1), which encodes a small protein hypothesized to be an extracellular ligand of EXS/EMS1. Here we show that rice contains two TPD1-like genes, OsTDL1A and OsTDL1B. Both are co-expressed with MSP1 in anthers during meiosis, but only OsTDL1A and MSP1 are co-expressed in the ovule. OsTDL1A binds to the leucine-rich-repeat domain of MSP1 in yeast two-hybrid assays and bimolecular fluorescence complementation in onion cells; OsTDL1B lacks this capacity. When driven by the maize Ubiquitin1 promoter, RNA interference against OsTDL1A phenocopies msp1 in the ovule but not in the anther. Thus, RNAi produces multiple MeMCs without causing male sterility. We conclude that OsTDL1A binds MSP1 in order to limit sporocyte numbers. OsTDL1A-RNAi lines may be suitable starting points for achieving synthetic apospory in rice.