[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.
[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 01/2013; 8(10):e76977. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: In microarray experiments, the global and the specific gene expression in the two-way table of gene x treatments (or tissues)
can be studied using linear-bilinear models that incorporate the main effects of genes (G), treatment (T), and gene x treatment
interaction (G x T). The plot of the first two axes obtained from the singular value decomposition of the bilinear (multiplicative)
term of these models (biplot) facilitates the interpretation of the gene expression patterns. In this study, two microarray
datasets were used to illustrate how two linear-bilinear models, the additive main effect and multiplicative interaction (AMMI)
and the treatment regression model (TREG) and their biplots can be used to determine the overall gene expression pattern across
treatments (or tissues) and for specific treatments. Dataset 1 had 5,339 genes and the objective was to identify genes with
modified expression during maize (Zea mays) seed development in response to different parental ploidy levels. In Dataset 2, the aim was to study gene expression in
15 tissue samples with different levels of development of breast cancer when compared with the expression of the genes in
noninfected tissues. The results from the analyses of Dataset 1 showed that the biplots of the AMMI and TREG models allow
identification of subsets of genes and treatments with noncrossover G x T interaction or with important levels of crossover
G x T. Results from Dataset 2 showed that the TREG model and its biplot facilitates the identification of genes with high
expression in all tumor cells. Also, the TREG biplots allowed identification of subsets of genes with a low level of gene
x tissue crossover interaction.
Journal of Agricultural Biological and Environmental Statistics 04/2012; 10(3):337-353. · 1.24 Impact Factor
[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.
Journal of Experimental Botany 03/2012; 63(10):3829-42. · 5.24 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 01/2012; 15(5):549 - 555. · 8.46 Impact Factor
[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. · 2.07 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: In Angiosperms, the male gametes are delivered to the female gametes through the maternal reproductive tissue by the pollen tube. Upon arrival, the pollen tube releases the two sperm cells, permitting double fertilization to take place. Although the critical role of the female gametophyte in pollen tube reception has been demonstrated, the underlying mechanisms remain poorly understood. Here, we describe lorelei, an Arabidopsis thaliana mutant impaired in sperm cell release, reminiscent of the feronia/sirène mutant. Pollen tubes reaching lorelei embryo sacs frequently do not rupture but continue to grow in the embryo sac. Furthermore, lorelei embryo sacs continue to attract additional pollen tubes after arrival of the initial pollen tube. The LORELEI gene is expressed in the synergid cells prior to fertilization and encodes a small plant-specific putative glucosylphosphatidylinositol-anchored protein (GAP). These results provide support for the concept of signaling mechanisms at the synergid cell membrane by which the female gametophyte recognizes the arrival of a compatible pollen tube and promotes sperm release. Although GAPs have previously been shown to play critical roles in initiation of fertilization in mammals, flowering plants appear to have independently evolved reproductive mechanisms that use the unique features of these proteins within a similar biological context.
The Plant Cell 12/2008; 20(11):3038-49. · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have further characterized floral organ-localized gene expression in the inflorescence of Arabidopsis thaliana by comparison of massively parallel signature sequencing (MPSS) data. Six libraries of RNA sequence tags from immature inflorescence tissues were constructed and matched to their respective loci in the annotated Arabidopsis genome. These signature libraries survey the floral transcriptome of wild-type tissue as well as the floral homeotic mutants, apetala1, apetala3, agamous, a superman/apetala1 double mutant, and differentiated ovules dissected from the gynoecia of wild-type inflorescences. Comparing and contrasting these MPSS floral expression libraries enabled demarcation of transcripts enriched in the petals, stamens, stigma-style, gynoecia, and those with predicted enrichment within the sepal/sepal-petals, petal-stamens, or gynoecia-stamens.
By comparison of expression libraries, a total of 572 genes were found to have organ-enriched expression within the inflorescence. The bulk of characterized organ-enriched transcript diversity was noted in the gynoecia and stamens, whereas fewer genes demonstrated sepal or petal-localized expression. Validation of the computational analyses was performed by comparison with previously published expression data, in situ hybridizations, promoter-reporter fusions, and reverse transcription PCR. A number of well-characterized genes were accurately delineated within our system of transcript filtration. Moreover, empirical validations confirm MPSS predictions for several genes with previously uncharacterized expression patterns.
This extensive MPSS analysis confirms and supplements prior microarray floral expression studies and illustrates the utility of sequence survey-based expression analysis in functional genomics. Spatial floral expression data accrued by MPSS and similar methods will be advantageous in the elucidation of more comprehensive genetic regulatory networks governing floral development.
[show abstract][hide abstract] ABSTRACT: Although many miRNAs are deeply conserved within each kingdom, none are known to be conserved between plants and animals. We identified Arabidopsis thaliana miR854 and miR855, two microRNAs (miRNAs) with multiple binding sites in the 3' untranslated region (3'UTR) of OLIGOURIDYLATE binding PROTEIN1b (At UBP1b), forming miRNA:mRNA interactions similar to those that cause translational repression/mRNA cleavage in animals. At UBP1b encodes a member of a heterogeneous nuclear RNA binding protein (hnRNP) family. The 3'UTR of At UBP1b is sufficient to repress reporter protein expression in tissues expressing miR854 or miR855 (rosette leaves and flowers, respectively) but not where both miRNAs are absent (cauline leaves). Intergenic regions containing sequences closely resembling miR854 are predicted to fold into stable miRNA precursors in animals, and members of the miR854 family are expressed in Caenorhabditis elegans, Mus musculus, and Homo sapiens, all with imperfect binding sites in the 3'UTR of genes encoding the T cell Intracellular Antigen-Related protein, an hnRNP of the UBP1 family. Potential binding sites for miR854 are absent from UBP1-like genes in fungi lacking the miRNA biogenetic machinery. Our results indicate that plants and animals share miRNAs of the miR854 family, suggesting a common origin of these miRNAs as regulators of basal transcriptional mechanisms.
The Plant Cell 01/2007; 18(12):3355-69. · 9.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: The WUSCHEL HOMEOBOX (WOX) gene family of Arabidopsis thaliana transcription factors is composed of at least 15 members encoding homeodomain proteins. We have characterized tepitzin1 (tpz1), a mutant harboring a transposon insertion in the 5′ regulatory region of WOX5. Heterozygous tpz1 plants do not have defects in vegetative growth or female reproductive development but exhibit aberrant pollen transmission, indicating that this mutation acts at the gametophytic level. WOX5 is abundantly expressed in mature pollen grains and elongating pollen tubes. Expression of WOX5 in heterozygous tpz1/+ anthers corresponds to 66% of WOX5 expression in wild-type anthers, confirming that WOX5 activity is affected in the mutant. WOX5-deficient pollen grains did not show developmental abnormalities and germinated at a frequency similar to wild-type pollen; however, in vitro and in vivo germination of pollen from tpz1 heterozygous individuals produced pollen tubes showing delayed germination and reduced length, confirming that the male gametophytic expression of WOX5 is necessary for normal pollen tube elongation. The reduction in average pollen tube length produced by the absence of WOX5 activity is less severe when heterozygous tpz1 individuals are in a homozygous quartet1 background, indicating that the absence of QRT1 activity partially compensates WOX5-deficient pollen tube elongation. Taken together, these results indicate that WOX5 activity is required for normal pollen tube growth in Arabidopsis. Our findings suggest that WOX5 identifies a new genetic pathway integrating homeobox transcription factors and auxin-mediated signals with the progamic phase of plant reproduction.
Sexual Plant Reproduction 01/2006; 19(4):163-173. · 2.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Chromatin-remodeling factors regulate the establishment of transcriptional programs during plant development. Although 42 genes encoding members of the SWI2/SNF2 family have been identified in Arabidopsis thaliana, <10 have been assigned a precise function on the basis of a mutant phenotype, and none have been shown to play a specific role during the gametophytic phase of the plant life cycle. A. thaliana chromatin-remodeling protein 11 (CHR11) encodes an imitation of switch (ISWI)-like chromatin-remodeling protein abundantly expressed during female gametogenesis and embryogenesis in Arabidopsis. To determine the function of CHR11 in wild-type plants, we introduced a hairpin construct leading to the production of double-stranded RNA, which specifically degraded the endogenous CHR11 mRNA by RNA interference (RNAi). Transcription of the RNAi-inducing hairpin RNA was driven by either a constitutive cauliflower mosaic virus 35S promoter (CaMV35S) acting at most stages of the sporophytic phase or a newly identified specific promoter acting at the onset of the female gametophytic phase (pFM1). All adult transformants that constitutively lacked sporophytic CHR11 activity showed reduced plant height and small cotyledonary embryos with limited cell expansion. In contrast, RNAi lines in which CHR11 was specifically silenced at the onset of female gametogenesis (megagametogenesis) had normal height and embryo size but had defective female gametophytes arrested before the completion of the mitotic haploid nuclear divisions. These results show that CHR11 is essential for haploid nuclear proliferation during megagametogenesis and cell expansion during the sporophytic phase, demonstrating the functional versatility of SWI2/SNF2 chromatin-remodeling factors during both generations of the plant life cycle.
Proceedings of the National Academy of Sciences 11/2005; 102(47):17231-6. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Genomic imprinting is a mitotically stable epigenetic modification that results in the functional non-equivalency of both parental genomes following fertilization. In flowering plants, studies of parent-of-origin effects have mostly identified genes that are only transcribed from a maternally inherited allele. In Arabidopsis, the Polycomb group protein MEDEA regulates seed development through the expression of the MADS-box gene PHERES1. Activation of the maternal MEDEA allele requires the function of DEMETER, a plant DNA glycosylase that also controls the transcriptional activity of the maternally inherited allele of the late-flowering gene FWA. Current studies of parent-of-origin effects have mostly identified genes that are only transcribed from a maternally inherited allele. Our current understanding of parent-of-origin effects could represent a new form of an Oedipus complex in which flowering plants prefer to rely transcriptionally on their maternal rather than their paternal chromosomes to ensure normal initiation of seed development.
Current Opinion in Plant Biology 03/2005; 8(1):19-25. · 8.46 Impact Factor