[Show abstract][Hide abstract] ABSTRACT: Here we describe a whole-mount immunolocalization protocol to follow the subcellular localization of proteins during female meiosis in Arabidopsis thaliana, a model species that is used to study sexual reproduction in flowering plants. By using confocal microscopy, the procedure allows one to follow megasporogenesis at all stages before differentiation of the functional megaspore. This in particular includes stages that occur during prophase I, such as the installation of the axial and central elements of the synaptonemal complex along the meiotic chromosomes. In contrast to procedures that require microtome sectioning or enzymatic isolation and smearing to separate female meiocytes from neighboring cells, this 3-day protocol preserves the constitution of the developing primordium and incorporates the architecture of the ovule to provide a temporal and spatial context to meiotic divisions. This opens up the possibility to systematically compare the dynamics of protein localization during female and mal
[Show abstract][Hide abstract] ABSTRACT: Viroids are small, covalently closed, circular non-coding RNA pathogens of flowering plants. It is proposed that the symptoms of viroid pathogenesis result from a direct interaction between the viroid genomic RNA and unknown host plant factors. Using a comparative genomic approach we took advantage of the detailed annotation of the Arabidopsis thaliana (Arabidopsis) genome to identify sequence homologies between putative viroid-derived small RNAs (vd-sRNAs) and coding regions in the plant genome. A pool of sequence homologies among 29 species of the Pospiviroidae family and the Arabidopsis genome was analyzed. Using this strategy we identified putative host gene targets that may be involved in symptom expression in viroid-infected plants. In this communication, we report the in silico prediction and the experimental validation of pospiviroid-derived sRNAs conserved in the lower strand of the pathogenicity domain of seven viroid species infecting tomato; those vd-sRNAs targeted for cleavage the host mRNA encoding a conserved tomato WD40-repeat protein (SolWD40-repeat; SGN_U563134). Analysis of SolWD40-repeat expression indicated that this gene is down-regulated in tomato plants infected with tomato planta macho viroid (TPMVd). Furthermore, 5′ RLM-RACE revealed that the SolWD40-repeat mRNA is cleaved at the predicted target site showing complementarity to a corresponding TPMVd-sRNA identified in silico. Our approach proved to be useful for the identification of natural host genes containing sequence homologies with segments of the Pospiviroidae genome. Using this strategy we identified a functionally conserved gene in Arabidopsis and tomato, whose expression was modified during viroid infection in the host genome; regulation of this gene expression could be guided by vd-sRNA:mRNA complementarity, suggesting that the comparison of the Arabidopsis genome to viroid sequences could lead to the identification of unexpected interactions between viroid RNAs and their host.
[Show abstract][Hide abstract] ABSTRACT: Key message
Meiosis and unreduced gametes.
Sexual flowering plants produce meiotically derived cells that give rise to the male and female haploid gametophytic phase. In the ovule, usually a single precursor (the megaspore mother cell) undergoes meiosis to form four haploid megaspores; however, numerous mutants result in the formation of unreduced gametes, sometimes showing female specificity, a phenomenon reminiscent of the initiation of gametophytic apomixis. Here, we review the developmental events that occur during female meiosis and megasporogenesis at the light of current possibilities to engineer unreduced gamete formation. We also provide an overview of the current understanding of mechanisms leading to parthenogenesis and discuss some of the conceptual implications for attempting the induction of clonal seed production in cultivated plants.
[Show abstract][Hide abstract] ABSTRACT: To investigate the genetic and molecular regulation that the female gametophyte could exert over neighboring sporophytic regions of the ovule, we performed a quantitative comparison of global expression in wild-type and nozzle/sporocyteless (spl) ovules of Arabidopsis thaliana (Arabidopsis), using Massively Parallel Signature Sequencing (MPSS). This comparison resulted in 1517 genes showing at least 3-fold increased expression in ovules lacking a female gametophyte, including those encoding 89 transcription factors, 50 kinases, 25 proteins containing a RNA-recognition motif (RRM), and 20 WD40 repeat proteins. We confirmed that eleven of these genes are either preferentially expressed or exclusive of spl ovules lacking a female gametophyte as compared to wild-type, and showed that six are also upregulated in determinant infertile1 (dif1), a meiotic mutant affected in a REC8-like cohesin that is also devoided of female gametophytes. The sporophytic misexpression of IOREMPTE, a WD40/transducin repeat gene that is preferentially expressed in the L1 layer of spl ovules, caused the arrest of female gametogenesis after differentiation of a functional megaspore. Our results show that in Arabidopsis, the sporophytic-gametophytic cross talk includes a negative regulation of the female gametophyte over specific genes that are detrimental for its growth and development, demonstrating its potential to exert a repressive control over neighboring regions in the ovule.
PLoS ONE 10/2013; 8(10):e76977. DOI:10.1371/journal.pone.0076977 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Female gametogenesis in most flowering plants depends on the predetermined selection of a single meiotically derived cell, as the three other megaspores die without further division or differentiation. Although in Arabidopsis thaliana the formation of the functional megaspore (FM) is crucial for the establishment of the gametophytic generation, the mechanisms that determine the specification and fate of haploid cells remain unknown. Here, we show that the classical arabinogalactan protein 18 (AGP18) exerts an active regulation over the selection and survival of megaspores in Arabidopsis. During meiosis, AGP18 is expressed in integumentary cells located in the abaxial region of the ovule. Overexpression of AGP18 results in the abnormal maintenance of surviving megaspores that can acquire a FM identity but is not sufficient to induce FM differentiation before meiosis, indicating that AGP18 positively promotes the selection of viable megaspores. We also show that all four meiotically derived cells in the ovule of Arabidopsis are competent to differentiate into a gametic precursor and that the function of AGP18 is important for their selection and viability. Our results suggest an evolutionary role for arabinogalactan proteins in the acquisition of monospory and the developmental plasticity that is intrinsic to sexual reproduction in flowering plants.
The Plant Cell 04/2013; 25(4). DOI:10.1105/tpc.112.106237 · 9.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The characterization of biomolecules from ancient samples can shed otherwise unobtainable insights into the past. Despite the fundamental role of transcriptomal change in evolution, the potential of ancient RNA remains unexploited - perhaps due to dogma associated with the fragility of RNA. We hypothesize that seeds offer a plausible refuge for long-term RNA survival, due to the fundamental role of RNA during seed germination. Using RNA-Seq on cDNA synthesized from nucleic acid extracts, we validate this hypothesis through demonstration of partial transcriptomal recovery from two sources of ancient maize kernels. The results suggest that ancient seed transcriptomics may offer a powerful new tool with which to study plant domestication.
PLoS ONE 01/2013; 8(1):e50961. DOI:10.1371/journal.pone.0050961 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apomixis is a natural form of asexual reproduction through seeds that leads to viable offspring genetically identical to the mother plant. New evidence from sexual model species indicates that the regulation of female gametogenesis and seed formation is also directed by epigenetic mechanisms that are crucial to control events that distinguish sexuality from apomixis, with important implications for our understanding of the evolutionary forces that shape structural variation and diversity in plant reproduction.
Current Opinion in Plant Biology 11/2012; 15(5-5):549 - 555. DOI:10.1016/j.pbi.2012.09.005 · 7.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To explore the molecular mechanisms that prevail during the establishment of the arbuscular mycorrhiza symbiosis involving the genus Glomus, we transcriptionally analysed spores of Glomus intraradices BE3 during early hyphal growth. Among 458 transcripts initially identified as being expressed at presymbiotic stages, 20% of sequences had homology to previously characterized eukaryotic genes, 30% were homologous to fungal coding sequences, and 9% showed homology to previously characterized bacterial genes. Among them, GintPbr1a encodes a homolog to Phenazine Biosynthesis Regulator (Pbr) of Burkholderia cenocepacia, an pleiotropic regulatory protein that activates phenazine production through transcriptional activation of the protein D isochorismatase biosynthetic enzyme phzD (Ramos et al., 2010). Whereas GintPbr1a is expressed during the presymbiotic phase, the G. intraradices BE3 homolog of phzD (BGintphzD) is transcriptionally active at the time of the establishment of the arbuscular mycorrhizal symbiosis. DNA from isolated bacterial cultures found in spores of G. intraradices BE3 confirmed that both BGintPbr1a and BGintphzD are present in the genome of its potential endosymbionts. Taken together, our results indicate that spores of G. intraradices BE3 express bacterial phenazine biosynthetic genes at the onset of the fungal-plant symbiotic interaction.
[Show abstract][Hide abstract] ABSTRACT: The life cycle of flowering plants alternates between a predominant sporophytic (diploid) and an ephemeral gametophytic (haploid) generation that only occurs in reproductive organs. In Arabidopsis thaliana, the female gametophyte is deeply embedded within the ovule, complicating the study of the genetic and molecular interactions involved in the sporophytic to gametophytic transition. Massively parallel signature sequencing (MPSS) was used to conduct a quantitative large-scale transcriptional analysis of the fully differentiated Arabidopsis ovule prior to fertilization. The expression of 9775 genes was quantified in wild-type ovules, additionally detecting >2200 new transcripts mapping to antisense or intergenic regions. A quantitative comparison of global expression in wild-type and sporocyteless (spl) individuals resulted in 1301 genes showing 25-fold reduced or null activity in ovules lacking a female gametophyte, including those encoding 92 signalling proteins, 75 transcription factors, and 72 RNA-binding proteins not reported in previous studies based on microarray profiling. A combination of independent genetic and molecular strategies confirmed the differential expression of 28 of them, showing that they are either preferentially active in the female gametophyte, or dependent on the presence of a female gametophyte to be expressed in sporophytic cells of the ovule. Among 18 genes encoding pentatricopeptide-repeat proteins (PPRs) that show transcriptional activity in wild-type but not spl ovules, CIHUATEOTL (At4g38150) is specifically expressed in the female gametophyte and necessary for female gametogenesis. These results expand the nature of the transcriptional universe present in the ovule of Arabidopsis, and offer a large-scale quantitative reference of global expression for future genomic and developmental studies.
[Show abstract][Hide abstract] ABSTRACT: The development of second-generation sequencing technologies has greatly benefitted the field of ancient DNA (aDNA). Its application can be further exploited by the use of targeted capture-enrichment methods to overcome restrictions posed by low endogenous and contaminating DNA in ancient samples. We tested the performance of Agilent's SureSelect and Mycroarray's MySelect in-solution capture systems on Illumina sequencing libraries built from ancient maize to identify key factors influencing aDNA capture experiments. High levels of clonality as well as the presence of multiple-copy sequences in the capture targets led to biases in the data regardless of the capture method. Neither method consistently outperformed the other in terms of average target enrichment, and no obvious difference was observed either when two tiling designs were compared. In addition to demonstrating the plausibility of capturing aDNA from ancient plant material, our results also enable us to provide useful recommendations for those planning targeted-sequencing on aDNA.
[Show abstract][Hide abstract] ABSTRACT: In flowering plants, the formation of gametes depends on the differentiation of cellular precursors that divide meiotically before giving rise to a multicellular gametophyte. The establishment of this gametophytic phase presents an opportunity for natural selection to act on the haploid plant genome by means of epigenetic mechanisms that ensure a tight regulation of plant reproductive development. Despite this early acting selective pressure, there are numerous examples of naturally occurring developmental alternatives that suggest a flexible regulatory control of cell specification and subsequent gamete formation in flowering plants. In this review, we discuss recent findings indicating that epigenetic mechanisms related to the activity of small RNA pathways prevailing during ovule formation play an essential role in cell specification and genome integrity. We also compare these findings to small RNA pathways acting during gametogenesis in animals and discuss their implications for the understanding of the mechanisms that control the establishment of the female gametophytic lineage during both sexual reproduction and apomixis.
Sexual Plant Reproduction 06/2011; 24(2):137-47. DOI:10.1007/s00497-011-0166-z · 0.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Defining the contributions and interactions of paternal and maternal genomes during embryo development is critical to understand the fundamental processes involved in hybrid vigor, hybrid sterility, and reproductive isolation. To determine the parental contributions and their regulation during Arabidopsis embryogenesis, we combined deep-sequencing-based RNA profiling and genetic analyses. At the 2-4 cell stage there is a strong, genome-wide dominance of maternal transcripts, although transcripts are contributed by both parental genomes. At the globular stage the relative paternal contribution is higher, largely due to a gradual activation of the paternal genome. We identified two antagonistic maternal pathways that control these parental contributions. Paternal alleles are initially downregulated by the chromatin siRNA pathway, linked to DNA and histone methylation, whereas transcriptional activation requires maternal activity of the histone chaperone complex CAF1. Our results define maternal epigenetic pathways controlling the parental contributions in plant embryos, which are distinct from those regulating genomic imprinting.
[Show abstract][Hide abstract] ABSTRACT: Brassinosteroids (BRs) are steroid-like hormones essential for plant growth and development. The most active forms of brassinosteroids are Brassinolide (BL) and Castasterone (CS), which are catalyzed by members of the CYP85A family of cytochrome P450 monooxygenases. In Arabidopsis thaliana there are two CYP85A gene members: CYP85A1 and CYP85A2. Unlike CYP85A1, CYP85A2 mediates the conversion of CS to BL. In contrast to mutations in CYP85A2 that result in severe dwarfism, cyp85a1 mutants do not show any obvious morphological phenotype during vegetative or floral development. By analyzing large-scale transcriptional activity in the ovule of Arabidopsis thaliana (Arabidopsis), we determined that CYP85A1 is abundantly expressed in wild-type but not in sporocyteless (spl) ovules lacking a female gametophyte. Insertional T-DNA lines defective in the activity of CYP85A1 exhibit a semi-sterile phenotype, suggesting a role for the corresponding enzyme acting at the gametophytic level. The CYP85A1 mRNA is localized in the female gametophyte and its neighboring sporophytic cells; however, translational fusions of the CYP85A1 promoter to uidA (GUS) showed GUS expression restricted to the female gametophyte, suggesting that within the ovule the corresponding protein is mostly active in gametophytic cells. A cytological analysis of heterozygous cyp85a1/+ individuals showed that close to 50% of female gametophytes are arrested before the first nuclear mitotic division of the haploid functional megaspore. Our results indicate that BR biosynthesis is required for the initiation of megagametogenesis in Arabidopsis.
[Show abstract][Hide abstract] ABSTRACT: Small RNAs emerged over the last decade as key regulators in diverse biological processes in eukaryotic organisms. To identify and study small RNAs, good and efficient protocols are necessary to isolate them, which sometimes may be challenging due to the composition of specific tissues of certain plant species. Here we describe a simple and efficient method to isolate small RNAs from different plant species.
We developed a simple and efficient method to isolate small RNAs from different plant species by first comparing different total RNA extraction protocols, followed by streamlining the best one, finally resulting in a small RNA extraction method that has no need of first total RNA extraction and is not based on the commercially available TRIzol® Reagent or columns. This small RNA extraction method not only works well for plant tissues with high polysaccharide content, like cactus, agave, banana, and tomato, but also for plant species like Arabidopsis or tobacco. Furthermore, the obtained small RNA samples were successfully used in northern blot assays.
Here we provide a simple and efficient method to isolate small RNAs from different plant species, such as cactus, agave, banana, tomato, Arabidopsis, and tobacco, and the small RNAs from this simplified and low cost method is suitable for downstream handling like northern blot assays.
[Show abstract][Hide abstract] ABSTRACT: Recent evidence indicates that the establishment of the haploid phase of the plant life cycle requires epigenetic mechanisms that control reproductive cell fate. We previously showed that in Arabidopsis thaliana (Arabidopsis) mutations in ARGONAUTE9 (AGO9) result in defective cell specification during megasporogenesis. AGO9 preferentially interacts with 24 nucleotide (nt) small RNAs (sRNAs) derived from transposable elements (TEs), and its sporophytic activity is required to silence TEs in the female gametophyte. Here we show that AGO9 can bind in vitro to 24 nt sRNAs corresponding to Athila retrotransposons expressed in the ovule prior to pollination. We also show that AGO9 is necessary to inactivate a significant proportion of long terminal repeat retrotransposons (LTRs) in the ovule, and that its predominant TE targets are located in the pericentromeric regions of all 5 chromosomes, suggesting a link between the AGO9-dependent sRNA pathway and heterochromatin formation. Our extended results point towards the existence of a tissue-specific mechanism of sRNA-dependent TE silencing in the ovule.
[Show abstract][Hide abstract] ABSTRACT: In the ovules of most sexual flowering plants female gametogenesis is initiated from a single surviving gametic cell, the functional megaspore, formed after meiosis of the somatically derived megaspore mother cell (MMC). Because some mutants and certain sexual species exhibit more than one MMC, and many others are able to form gametes without meiosis (by apomixis), it has been suggested that somatic cells in the ovule are competent to respond to a local signal likely to have an important function in determination. Here we show that the Arabidopsis protein ARGONAUTE 9 (AGO9) controls female gamete formation by restricting the specification of gametophyte precursors in a dosage-dependent, non-cell-autonomous manner. Mutations in AGO9 lead to the differentiation of multiple gametic cells that are able to initiate gametogenesis. The AGO9 protein is not expressed in the gamete lineage; instead, it is expressed in cytoplasmic foci of somatic companion cells. Mutations in SUPPRESSOR OF GENE SILENCING 3 and RNA-DEPENDENT RNA POLYMERASE 6 exhibit an identical defect to ago9 mutants, indicating that the movement of small RNA (sRNAs) silencing out of somatic companion cells is necessary for controlling the specification of gametic cells. AGO9 preferentially interacts with 24-nucleotide sRNAs derived from transposable elements (TEs), and its activity is necessary to silence TEs in female gametes and their accessory cells. Our results show that AGO9-dependent sRNA silencing is crucial to specify cell fate in the Arabidopsis ovule, and that epigenetic reprogramming in companion cells is necessary for sRNA-dependent silencing in plant gametes.
[Show abstract][Hide abstract] ABSTRACT: Maize domestication (Zea mays ssp. mays L.) resulted in a wide diversity of native landraces that represent an invaluable source of genetic information for exploring natural variation and genome evolution. We sequenced de novo the approximately 2-gigabase genome of the Mexican landrace Palomero Toluqueño (Palomero) and compared its features to those of the modern inbred line B73. We revealed differences concordant with its ancient origin and identified chromosomal regions of low nucleotide variability that contain domestication genes involved in heavy-metal detoxification. Our results indicate that environmental changes were important selective forces acting on maize domestication.
[Show abstract][Hide abstract] ABSTRACT: The domestication of maize gave rise to a group of ancestral landraces that eventually diversified and adapted to a wide range
of climatic and geographic conditions. Although biologists do not always agree in the total number of landraces currently
existing in Mexico, there are at least 59 that can be clearly and consistently distinguished on the basis of biochemical and
morphological characteristics. Following a historical perspective, this chapter reviews our current knowledge of the phenotypic
and geographical distinctions among Mexican landraces, and illustrates their most recent classification. It also discusses
some of the opportunities that the genomic characterization of landrace germplasm could offer for the study of maize functional
diversity and molecular evolution.