Showing papers in "Sexual Plant Reproduction in 2017"

Haploid embryo production in rice and maize induced by PsASGR - BBML transgenes

Abstract: The PsASGR - BBML transgene, derived from a wild apomictic grass species, can induce parthenogenesis, embryo formation without fertilization, in rice and maize, leading to the formation of haploid plants. The ability to engineer apomictic crop plants using genes identified from naturally occurring apomicts will depend on the ability of those genes to function in crop plants. The PsASGR-BBML transgene, derived from the apomictic species Pennisetum squamulatum, promotes parthenogenesis in sexual pearl millet, a member of the same genus, leading to the formation of haploid embryos. This study determined that the PsASGR-BBML transgene can induce haploid embryo development in two major monocot crops, maize and rice. Transgene variations tested included two different promoters and the use of both genomic and cDNA PsASGR-BBML-derived sequences. Haploid plants were recovered from mature caryopses (seed) of rice and maize lines at variable rates. The PsASGR-BBML transgenes failed to induce measurable haploid seed development in the model genetic plant system Arabidopsis thaliana. Complexity of embryo development, as documented in transgenic rice lines, identifies the need for further characterization of the PsASGR-BBML gene.

Untargeted metabolomic analysis of tomato pollen development and heat stress response

Abstract: Pollen development metabolomics Developing pollen is among the plant structures most sensitive to high temperatures, and a decrease in pollen viability is often associated with an alteration of metabolite content Most of the metabolic studies of pollen have focused on a specific group of compounds, which limits the identification of physiologically important metabolites To get a better insight into pollen development and the pollen heat stress response, we used a liquid chromatography–mass spectrometry platform to detect secondary metabolites in pollen of tomato (Solanum lycopersicum L) at three developmental stages under control conditions and after a short heat stress at 38 °C Under control conditions, the young microspores accumulated a large amount of alkaloids and polyamines, whereas the mature pollen strongly accumulated flavonoids The heat stress treatment led to accumulation of flavonoids in the microspore The biological role of the detected metabolites is discussed This study provides the first untargeted metabolomic analysis of developing pollen under a changing environment that can serve as reference for further studies

Characterization of pollen-expressed bZIP protein interactions and the role of ATbZIP18 in the male gametophyte.

Abstract: KEY MESSAGE : bZIP TF network in pollen. Transcriptional control of gene expression represents an important mechanism guiding organisms through developmental processes and providing plasticity towards environmental stimuli. Because of their sessile nature, plants require effective gene regulation for rapid response to variation in environmental and developmental conditions. Transcription factors (TFs) provide such control ensuring correct gene expression in spatial and temporal manner. Our work reports the interaction network of six bZIP TFs expressed in Arabidopsis thaliana pollen and highlights the potential functional role for AtbZIP18 in pollen. AtbZIP18 was shown to interact with three other pollen-expressed bZIP TFs-AtbZIP34, AtbZIP52, and AtbZIP61 in yeast two-hybrid assays. AtbZIP18 transcripts are highly expressed in pollen, and at the subcellular level, an AtbZIP18-GFP fusion protein was located in the nucleus and cytoplasm/ER. To address the role of AtbZIP18 in the male gametophyte, we performed phenotypic analysis of a T-DNA knockout allele, which showed slightly reduced transmission through the male gametophyte. Some of the phenotype defects in atbzip18 pollen, although observed at low penetrance, were similar to those seen at higher frequency in the T-DNA knockout of the interacting partner, AtbZIP34. To gain deeper insight into the regulatory role of AtbZIP18, we analysed atbzip18/- pollen microarray data. Our results point towards a potential repressive role for AtbZIP18 and its functional redundancy with AtbZIP34 in pollen.

Sequencing the genomic regions flanking S-linked PvGLO sequences confirms the presence of two GLO loci, one of which lies adjacent to the style-length determinant gene CYP734A50

Abstract: Primula vulgaris contains two GLOBOSA loci, one located adjacent to the style length determinant gene CYP734A50 which lies within the S -locus. Using a combination of BAC walking and PacBio sequencing, we have sequenced two substantial genomic contigs in and around the S-locus of Primula vulgaris. Using these data, we were able to demonstrate that two alleles of PvGlo P as well as PvGlo T can be present in the genome of a single plant, providing empirical evidence that these two forms of the MADS-box gene GLOBOSA are separate loci and not allelic as previously reported. We propose they should be renamed PvGLO1 and PvGLO2. BAC contigs extending from each GLOBOSA locus were identified and fully sequenced. No homologous genes were found between the contigs other than the GLOBOSA genes themselves, consistent with their identity as separate loci. Exons of the recently identified style-length determinant gene CYP734A50 were identified on one end of the contig containing PvGLO2 and these genes are adjacent in the genome, suggesting that PvGLO2 lies either within or at least very close to the S-locus. Current evidence suggests that both CYP734A50 and GLO2 are specific to the S-morph mating type and are hemizygous rather than heterozygous in the Primula genome. This finding contrasts classical models of the HSI locus, which propose that components of the S-locus are allelic, suggesting that these models may need to be reconsidered.

Tracking maize pollen development by the Leaf Collar Method.

Abstract: An easy and highly reproducible nondestructive method named the Leaf Collar Method is described to identify and characterize the different stages of pollen development in maize. In plants, many cellular events such as meiosis, asymmetric cell division, cell cycle regulation, cell fate determination, nucleus movement, vacuole formation, chromatin condensation and epigenetic modifications take place during pollen development. In maize, pollen development occurs in tassels that are confined within the internal stalk of the plant. Hence, identification of the different pollen developmental stages as a tool to investigate above biological processes is impossible without dissecting the entire plant. Therefore, an efficient and reproducible method is necessary to isolate homogeneous cell populations at individual stages throughout pollen development without destroying the plant. Here, we describe a method to identify the various stages of pollen development in maize. Using the Leaf Collar Method in the maize inbreed line B73, we have determined the duration of each stage from pollen mother cells before meiosis to mature tricellular pollen. Anther and tassel size as well as percentage of pollen stages were correlated with vegetative stages, which are easily recognized. The identification of stage-specific genes indicates the reproducibility of the method. In summary, we present an easy and highly reproducible nondestructive method to identify and characterize the different stages of pollen development in maize. This method now opens the way for many subsequent physiological, morphological and molecular analyses to study, for instance, transcriptomics, metabolomics, DNA methylation and chromatin patterns during normal and stressful conditions throughout pollen development in one of the economically most important grass species.

Molecular movement in the Arabidopsis thaliana female gametophyte

Abstract: Size limits on molecular movement among female gametes. Cellular decisions can be influenced by information communicated from neighboring cells. Communication can occur via signaling or through the direct transfer of molecules. Movement of RNAs and proteins has frequently been observed among symplastically connected plant cells. In flowering plants, the female gametes, the egg cell and central cell, are closely apposed within the female gametophyte. Here we investigated the ability of fluorescently labeled dyes and small RNAs to move from the Arabidopsis thaliana central cell to the egg apparatus following microinjection. These results define a size limit of at least 20 kDa for symplastic movement between the two gametes, somewhat larger than that previously observed in Torenia fournieri. Our results indicate that symplastic connectivity in Arabidopsis thaliana changes after fertilization and suggest that prior to fertilization mechanisms are in place to facilitate small RNA movement from the central cell to the egg cell and synergids.

Advances in understanding the molecular mechanisms of cytoplasmic male sterility and restoration in rice

Abstract: Cytoplasmic male sterility (CMS) in plants is a male reproductive defect determined by mitochondrial genes and inherited maternally. CMS can be suppressed by nuclear restorer of fertility (Rf) genes. Therefore, CMS/Rf systems provide a classic model for the study of mitochondrial–nuclear interactions in plants. Moreover, CMS/Rf systems are economical, effective tools for the production of hybrid seeds. For example, CMS/Rf systems have been applied in over forty countries to breed hybrid rice (Oryza sativa L.) with improved yields due to hybrid vigor. The production of hybrid rice mainly depends on three types of CMS systems, namely Wild-Abortive type CMS (CMS-WA), Hong-Lian type CMS (CMS-HL) and Boro II type CMS (CMS-BT). Understanding the molecular mechanisms underlying these CMS/Rf systems will help us to understand mitochondrial–nuclear interactions, and accelerate the utilization of heterosis for improvement in yield. In the past decades, research benefitting from the availability of the high-quality, annotated mitochondrial and nuclear genome sequences of rice has isolated many CMS genes, identified the cognate nuclear Rf genes and studied the molecular mechanisms underlying CMS and restoration in rice. Here, we focus on recent advances in studies of the three major CMS/Rf systems in rice and discuss the key issues facing basic research and application of CMS/Rf systems in the future.

Dynamics of post-translationally modified histones during barley pollen embryogenesis in the presence or absence of the epi-drug trichostatin A

Abstract: Improving pollen embryogenesis. Despite the agro-economic importance of pollen embryogenesis, the mechanisms underlying this process are still poorly understood. We describe the dynamics of chromatin modifications (histones H3K4me2, H3K9ac, H3K9me2, and H3K27me3) and chromatin marks (RNA polymerase II CDC phospho-Ser5, and CENH3) during barley pollen embryogenesis. Immunolabeling results show that, in reaction to stress, immature pollen rapidly starts reorganizing several important chromatin modifications indicative of a change in cell fate. This new chromatin modification pattern was accomplished within 24 h from whereon it remained unaltered during subsequent mitotic activity. This indicates that cell fate transition, the central element of pollen embryogenesis, is completed early on during the induction process. Application of the histone deacetylase inhibitor trichostatin A stimulated pollen embryogenesis when used on pollen with a gametophytic style chromatin pattern. However, when this drug was administered to embryogenic pollen, the chromatin markers reversed toward a gametophytic profile, embryogenesis was halted and all pollen invariably died.

Calcium controls the formation of vacuoles from mitochondria to regulate microspore development in wheat.

Abstract: Potassium antimonite was used to investigate the localisation of calcium in developing wheat anthers to examine the relationship between Ca2+ and pollen development. During anther development, calcium precipitate formation increased in anther wall cells prior to microspore mother cell meiosis and appeared in microspores, suggesting the presence of a calcium influx from anther wall cells into the locule. Initially, the precipitates in microspore cytoplasm primarily accumulated in the mitochondria and destroyed their inner membranes (cisterns) to become small vacuoles, which expanded and fused, ultimately becoming a large vacuole during microspore vacuolisation. After microspore division and large vacuole decomposition, many calcium precipitates again accumulated in the small vacuoles, indicating that calcium from the large vacuole moved back into the cytoplasm of bicellular pollen.

Functional characterization of AGAMOUS-subfamily members from cotton during reproductive development and in response to plant hormones.

Abstract: Expression analysis of the AG -subfamily members from G. hirsutum during flower and fruit development. Reproductive development in cotton, including the fruit and fiber formation, is a complex process; it involves the coordinated action of gene expression regulators, and it is highly influenced by plant hormones. Several studies have reported the identification and expression of the transcription factor family MADS-box members in cotton ovules and fibers; however, their roles are still elusive during the reproductive development in cotton. In this study, we evaluated the expression profiles of five MADS-box genes (GhMADS3, GhMADS4, GhMADS5, GhMADS6 and GhMADS7) belonging to the AGAMOUS-subfamily in Gossypium hirsutum. Phylogenetic and protein sequence analyses were performed using diploid (G. arboreum, G. raimondii) and tetraploid (G. barbadense, G. hirsutum) cotton genomes, as well as the AG-subfamily members from Arabidopsis thaliana, Petunia hybrida and Antirrhinum majus. qPCR analysis showed that the AG-subfamily genes had high expression during flower and fruit development in G. hirsutum. In situ hybridization analysis also substantiates the involvement of AG-subfamily members on reproductive tissues of G. hirsutum, including ovule and ovary. The effect of plant hormones on AG-subfamily genes expression was verified in cotton fruits treated with gibberellin, auxin and brassinosteroid. All the genes were significantly regulated in response to auxin, whereas only GhMADS3, GhMADS4 and GhMADS7 genes were also regulated by brassinosteroid treatment. In addition, we have investigated the GhMADS3 and GhMADS4 overexpression effects in Arabidopsis plants. Interestingly, the transgenic plants from both cotton AG-like genes in Arabidopsis significantly altered the fruit size compared to the control plants. This alteration suggests that cotton AG-like genes might act regulating fruit formation. Our results demonstrate that members of the AG-subfamily in G. hirsutum present a conserved expression profile during flower development, but also demonstrate their expression during fruit development and in response to phytohormones.

Developmental landmarks during floral ontogeny of jalapeño chili pepper (Capsicum annuum L.) and the effect of gibberellin on ovary growth.

Abstract: Pepper (Capsicum annuum L.) is an important horticultural crop in many regions of the world. The final shape and size of the fruit are known to be determined at a very early step of flower development. During flower development hormonal treatments using gibberellins seem to promote growth resulting in higher yield and fruit quality. However, the morphological changes that occur in the pepper flowers after these treatments are largely unknown. In the present study, we provide a description of floral development landmarks of jalapeno chili pepper (cultivar Huichol), divided in nine representative stages from its initiation until the opening of the bud. We established a correlation among external flower development and the time and pattern of reproductive organogenesis. Male and female gametogenesis progression was used to define specific landmarks during flower maturation. The pattern of expression of key genes involved in gibberellin metabolism and response was also evaluated in the nine flower stages. The proposed development framework was used to analyze the effect of gibberellin treatments in the development of the flower. We observed both an effect of the treatment in the histology of the ovary tissue and an increase in the level of expression of CaGA2ox1 and CaGID1b genes. The developmental stages we defined for this species are very useful to analyze the molecular and morphological changes after hormonal treatments.

Arabidopsis thaliana MLO genes are expressed in discrete domains during reproductive development

Abstract: MLOs in Plant Reproduction. The MILDEW RESISTANCE LOCUS-O (MLO) protein family, comprised of 15 members, plays roles in diverse cell-cell communication processes such as powdery mildew susceptibility, root thigmomorphogenesis, and pollen tube reception. The NORTIA (NTA, AtMLO7) gene is expressed in the synergid cells of the female gametophyte where it functions in intercellular communication with the pollen tube. Discrepancies between previously published promoter::GUS and promoter::gene-GUS constructs expression patterns led us to explore the regulation of NTA expression. Here we found via NTApro::gNTA-GUS truncations that sequences within the NTA gene negatively regulate its expression in the stomata and carpel walls. This led to the hypothesis that other MLO family members may also have additional regulatory sequences within the gene. MLOpro::gMLO-GUS constructs were examined for each family member focusing specifically on flowers in order to determine whether other MLOs could play a role in reproductive cell-cell communication. Notably, several MLOs were expressed in the pollen, in the stigma, in the pollinated style, and in the synergids and central cell. These findings indicate that other MLOs in addition to NTA could play a role in reproduction. Previous studies on the MLO family showed that phylogenetically related MLOs had redundant functions in powdery mildew infection and root thigmomorphogenesis; however, MLO expression in reproductive tissues did not strictly follow phylogenetic relationships, indicating that MLOs from different evolutionary origins may have been recruited for function in sexual reproduction.

Style morphology and pollen tube pathway.

Abstract: The style morphology and anatomy vary among different species. Three basic types are: open, closed, and semi-closed. Cells involved in the pollen tube pathway in the different types of styles present abundant endoplasmic reticulum, dictyosomes, mitochondria, and ribosomes. These secretory characteristics are related to the secretion where pollen tube grows. This secretion can be represented by the substances either in the canal or in the intercellular matrix or in the cell wall. Most studies suggest that pollen tubes only grow through the secretion of the canal in open styles. However, some species present pollen tubes that penetrate the epithelial cells of the canal, or grow through the middle lamella between these cells and subepithelial cells. In species with a closed style, a pathway is provided by the presence of an extracellular matrix, or by the thickened cell walls of the stylar transmitting tissue. There are reports in some species where pollen tubes can also penetrate the transmitting tissue cells and continue their growth through the cell lumen. In this review, we define subtypes of styles according to the path of the pollen tube. Style types were mapped on an angiosperm phylogenetic tree following the maximum parsimony principle. In line with this, it could be hypothesized that: the open style appeared in the early divergent angiosperms; the closed type of style originated in Asparagales, Poales, and Eudicots; and the semi-closed style appeared in Rosids, Ericales, and Gentianales. The open style seems to have been lost in core Eudicots, with reversions in some Rosids and Asterids.

Evidence for post-zygotic self-incompatibility in Handroanthus impetiginosus (Bignoniaceae)

Abstract: Late-acting self-incompatibility (LSI) has been defined as a genetically controlled self-sterility mechanism that prevents seed set by selfing, despite normal pollen tube growth and ovule penetration in self-pollinated pistils. In species of the Bignoniaceae with LSI, such as Handroanthus impetiginosus, the selfed pistils are characterized by a marked delay in ovule penetration, fertilization, and endosperm initiation, followed by uniform pistil abscission. This highlights the contentious possibility of a post-zygotic self-incompatibility system. However, previous studies were unable to confirm fusion of the sperm and egg cell nuclei in selfed ovules. In the present study, the cytology of the embryo sac, double fertilization, and pistil longevity was investigated in H. impetiginosus using comparative nuclei microspectrofluorometry of DAPI-stained sections of self- vs. unpollinated pistils. Differences in both pistil longevity and ovary size between self- and unpollinated flowers at the time of pistil abscission were significant. Zygotes with double the DNA content in their nuclei relative to unfertilized egg cell nuclei were verified in selfed ovules from the first day after pollination onward, and G1 karyogamy appeared to have occurred. Our cytological analysis clearly indicates that ovules of self-pollinated pistils in H. impetiginosus are fertilized before pistil abscission but no embryogenesis initiation occurs, which strongly supports the idea of a post-zygotic self-incompatibility mechanism.

Loss of GCS1/HAP2 does not affect the ovule-targeting behavior of pollen tubes.

Abstract: hap2-1 pollen tube ovule targeting. Upon pollination, a pollen grain germinates to produce a pollen tube, which grows through the style to deliver two immobile sperm cells to the female gametophyte. Double fertilization is completed after the pollen tube enters an ovule. GENERATIVE CELL SPECIFIC 1 (GCS1)/HAPLESS 2 (HAP2) contributes to the fusion of gametes at fertilization and has been suggested to affect pollen tube guidance. However, there is controversy over the role of GCS1/HAP2 in pollen tube guidance because of conflicting results from different studies. To characterize the effects of the gcs1/hap2 mutation on pollen tube behavior, we analyzed the Arabidopsis thaliana hap2-1/HAP2 mutant, which carries a gcs1/hap2 mutation in the quartet background. The quartet mutant produces tetrads consisting of four pollen grains that remain adherent after the pollen mother cell has completed meiosis. Thus, a hap2-1/HAP2 tetrad contains hap2-1 and HAP2 pollen grains in a 2:2 ratio. Moreover, the hap2-1 locus is linked to the β-glucuronidase (GUS) gene. An excess pollination experiment with hap2-1/HAP2 tetrads revealed that the hap2-1 pollen tube targets ovules normally. Additionally, the results of restricted pollination and aniline blue staining indicated that there are no significant differences between the ovule-targeting frequencies of pollen tubes from hap2-1/HAP2 and HAP2/HAP2 tetrads.

Sex allocation in gynodioecious Cyananthus delavayi differs between gender morphs and soil quality.

Abstract: Sex allocation in Cyananthus delavayi. Gynodioecy, where females and hermaphrodites coexist in the same natural population, is particularly suitable for predicting the ecological pressures that drive the stability of gender polymorphism. Since females have a disadvantage in that they only contribute to the next generation via ovules, they should gain an advantage via other means, of which resource allocation is an important component. Thus, to study their sex allocation is very helpful to understand how the dimorphic sexual system is maintained in natural systems. We studied the sex allocation patterns and reproductive output of the gynodioecious Cyananthus delavayi in three populations with different soil qualities (organic matter, N, P and K). The hermaphroditic flowers and pistils were much larger than those of female individuals. Although both gender morphs invested similar biomass in the pistils, females allocated more of their resource pool to the seed production, while hermaphrodites allocated more to pollinator advertisement. The pollen production of hermaphrodites did not differ between populations, suggesting that pollen production by hermaphrodites was not limited by soil nutrients. Fruit set of females, but not hermaphrodites, decreased with declining soil quality, whereas seeds per fruit of both females and hermaphrodites were highest in poor soils. Overall, this study shows that females achieve greater reproductive success by allocating more of their resource pool to enhancing seed production, which should favor their presence in gynodioecious populations. The hermaphrodites achieve reproductive success from both pollen and seed production, and unnecessarily reduce their allocation to pollen production. Soil quality should explain, at least partially, the sexual allocation patterns. Furthermore, some of our findings contradict previous hypotheses, thus adding a new example to the body of research on plant sex allocation and the development of future theories.