-
[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; · 8.99 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The functional annotation of completely sequenced genomes requires fast and reliable procedures for the systematic determination
of in situ patterns of gene expression. Although several whole-mount hybridization protocols have been successfully used to
localize mRNAs in soft and easily dissected tissues such as roots, seedlings, and developing flowers, their use has been hampered
by the small size and inaccessibility of developing ovules and megagametophytes. We have implemented a whole-mount in situ
hybridization procedure that overcomes these difficulties inArabidopsis thaliana. Multiple developing gynoecia, mature ovules, and nascent seeds are dissected, fixed, and embedded on a soft layer of 15%
polyacrylamide before being processed for hybridization with digoxygeninlabeled RNA probes. We characterized the pattern of
expression of a gene encoding a UDP-D-glucuronate 4-epimerase (GAE5) and demonstrated that the procedure can be used to determine highly dynamic patterns of mRNA localization early during development
of ovules. We confirmed the reliability of our approach by detectinguidA (GUS) mRNA localization patterns in ovules from enhancer detector or transformant lines that showed glucuronidase (GUS) protein
localization in specific cells of the megagametophyte. The method can be successfully used to localize mRNA transcripts and
achieve single-cell resolution in both sporophytic and gametophytic cells at all stages of development of megagametophytes
and during seed initiation. It constitutes an initial step toward the automation of high-throughput in situ hybridization
procedures for functional studies of female reproductive development and early seed formation.
Plant Molecular Biology Reporter 04/2012; 23(3):279-289. · 2.45 Impact Factor
-
Nidia Sánchez-León,
Mario Arteaga-Vázquez,
César Alvarez-Mejía,
Javier Mendiola-Soto,
Noé Durán-Figueroa,
Daniel Rodríguez-Leal,
Isaac Rodríguez-Arévalo,
Vicenta García-Campayo,
Marcelina García-Aguilar,
Vianey Olmedo-Monfil,
Mario Arteaga-Sánchez,
Octavio Martínez de la Vega,
Kan Nobuta,
Kalyan Vemaraju,
Blake C Meyers, Jean-Philippe Vielle-Calzada
[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. · 5.36 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. · 1.87 Impact Factor
-
Daphné Autran,
Célia Baroux,
Michael T Raissig,
Thomas Lenormand,
Michael Wittig,
Stefan Grob,
Andrea Steimer,
Matthias Barann,
Ulrich C Klostermeier,
Olivier Leblanc, Jean-Philippe Vielle-Calzada,
Phillip Rosenstiel,
Daniel Grimanelli,
Ueli Grossniklaus
[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.
Cell 05/2011; 145(5):707-19. · 32.40 Impact Factor
-
[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.
Plant signaling & behavior 03/2011; 6(3):321-6.
-
[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.
Plant Methods 02/2011; 7:4. · 2.83 Impact Factor
-
[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.
Plant signaling & behavior 11/2010; 5(11):1476-9.
-
[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.
Nature 03/2010; 464(7288):628-32. · 36.28 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Whether deposited maternal products are important during early seed development in flowering plants remains controversial. Here, we show that RNA interference-mediated downregulation of transcription is deleterious to endosperm development but does not block zygotic divisions. Furthermore, we show that RNA POLYMERASE II is less active in the embryo than in the endosperm. This dimorphic pattern is established late during female gametogenesis and is inherited by the two products of fertilization. This juxtaposition of distinct transcriptional activities correlates with differential patterns of histone H3 lysine 9 dimethylation, LIKE HETEROCHROMATIN PROTEIN1 localization, and Histone H2B turnover in the egg cell versus the central cell. Thus, distinct epigenetic and transcriptional patterns in the embryo and endosperm are already established in their gametic progenitors. We further demonstrate that the non-CG DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and DEMETER-LIKE DNA glycosylases are required for the correct distribution of H3K9 dimethylation in the egg and central cells, respectively, and that plants defective for CMT3 activity show abnormal embryo development. Our results provide evidence that cell-specific mechanisms lead to the differentiation of epigenetically distinct female gametes in Arabidopsis thaliana. They also suggest that the establishment of a quiescent state in the zygote may play a role in the reprogramming of the young plant embryo.
The Plant Cell 02/2010; 22(2):307-20. · 8.99 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.
BMC Plant Biology 02/2008; 8:43. · 3.45 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Abstract
Background
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.
Results
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.
Conclusion
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.
BMC Plant Biology. 01/2008;
-
[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. · 8.99 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. · 9.68 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. · 9.27 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Classical arabinogalactan proteins (AGPs) are an abundant class of cell surface proteoglycans widely distributed in flowering plants. We have used a combination of enhancer detection tagging and RNA interference (RNAi)-induced posttrancriptional silencing to demonstrate that AGP18, a gene encoding a classical arabinogalactan protein, is essential for female gametogenesis in Arabidopsis thaliana. AGP18 is expressed in cells that spatially and temporally define the sporophytic to gametophytic transition and during early stages of seed development. More than 75% of the T1 transformants resulted in T2 lines showing reduced seed set during at least three consecutive generations but no additional developmental defects. AGP18-silenced T2 lines showed reduced AGP18 transcript levels in female reproductive organs, the presence of 21-bp RNA fragments specific to the AGP18 gene, and the absence of in situ AGP18 mRNA localization in developing ovules. Reciprocal crosses to wild-type plants indicate that the defect is female specific. The genetic and molecular analysis of AGP18-silenced plants containing a single T-DNA RNAi insertion suggests that posttranscriptional silencing of AGP18 is acting both at the sporophytic and gametophytic levels. A cytological analysis of all defective AGP18-RNAi lines, combined with the analysis of molecular markers acting at key stages of female gametogenesis, showed that the functional megaspore fails to enlarge and mitotically divide, indicating that AGP18 is essential to initiate female gametogenesis in Arabidopsis. Our results assign a specific function in plant development to a gene encoding a classical AGP.
The Plant Cell 11/2004; 16(10):2614-28. · 8.99 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Although more than 25,000 genes of Arabidopsis thaliana have been sequenced and mapped, adequate expression or functional information is available for less than 15% of them. In the case of Oryza sativa (rice), about half of more than 55,000 predicted genes have been assigned to a vague functional category on the basis of their sequence, but fewer than 100 have been ascribed a precise, verified function after the identification of a mutant phenotype caused by the molecular disruption of the corresponding gene. Enhancer detection and gene trapping represent insertional mutagenesis strategies that report random expression of many genes and often generate loss-of-function mutations. Several trapping vectors have been designed in a limited number of species, and large-scale enhancer detection and gene trap screens that aim to generate a wide range of spatially and temporally restricted expression patterns have been initiated in both Arabidopsis and rice. These strategies are proving to be essential to the functional annotation of completely sequenced genomes, enabling the analysis of gene function in the context of the entire plant life cycle and substantially expanding our understanding of plant growth and development.
Methods in molecular biology (Clifton, N.J.) 02/2004; 267:397-414.
-
[show abstract]
[hide abstract]
ABSTRACT: The spindle plays a central role in chromosome segregation during mitosis and meiosis. In particular, various kinesins are thought to play crucial roles in spindle structure and function in both mitosis and meiosis of fungi and animals. A group of putative kinesins has been previously identified in Arabidopsis, called ATK1-ATK4 (previously known as KATA-KATD), but their in vivo functions have not been tested with genetic studies. We report here the isolation and characterization of a mutant, atk1-1, which has a defective ATK1 gene. The atk1-1 mutant was identified in a collection of Ds transposon insertion lines by its reduced fertility. Reciprocal crosses between the atk1-1 mutant and wild type showed that only male fertility was reduced, not female fertility. Molecular analyses, including revertant studies, indicated that the Ds insertion in the ATK1 gene was responsible for the fertility defect. Light microscopy revealed that, in the atk1-1 mutant, male meiosis was defective, producing an abnormal number of microspores of variable sizes. Further cytological studies indicated that meiotic chromosome segregation and spindle organization were both abnormal in the mutant. Specifically, the atk1-1 mutant male meiotic cells had spindles that were broad, unfocused and multi-axial at the poles at metaphase I, unlike the typical fusiform bipolar spindle found in the wild-type metaphase I cells. Therefore, the ATK1 gene plays a crucial role in spindle morphogenesis in male Arabidopsis meiosis.
Development 06/2002; 129(10):2401-9. · 6.60 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The reproductive system determines the way in which gametes develop and interact to form a new organism. Therefore, it exerts
the primary level of control of genotypic frequencies in plant populations, and plays a fundamental role in plant breeding.
A basic understanding of plant reproductive development will completely transform current breeding strategies used for seed
production. Apomixis is an asexual form of reproduction in which embryogenesis occurs in a cell lineage lacking both meiosis
and fertilization, and that culminates in the formation of viable progeny genetically identical to the mother plant. The transfer
of apomixis into sexual crops will allow the production of self-perpetuating improved hybrids, and the fixation of any desired
heterozygous genotype. The initiation of apomictic development invariably takes place at early stages of ovule ontogeny, before
the establishment of the megagametophytic phase. The developmental versatility associated with megagametophyte formation suggests
that the genetic and molecular regulation of apomixis is intimately related to the regulation of sexuality. Differences between
the initiation of sexual and apomictic development may be determined by regulatory genes that act during megasporogenesis,
and that control events leading to the formation of unreduced female gametophytes. To test this hypothesis, we are isolating
and characterizing genes that act during megasporogenesis inArabidopsis thaliana and investigating their potential role in the induction of apomixis. We are using a recently established transposon-based
enhancer detection and gene trap insertional mutagenesis system that allows the identification of genes based on their expression
patterns. An initial screen of transposants has yielded over 20 lines conferring restricted GUS expression during early ovule
development. We have obtained the sequence of genomic fragments flanking the transposon insertion. Several have homology to
genes playing important roles in plant and animal development. They include cell cycle regulators, enzymes involved in callose
hydrolysis, leucine-rich repeat protein kinase receptors, and expressed sequence tags (ESTs) of unknown function. Independently,
a genetic screen allows the identification of female sterile mutants defective in megasporogenesis. Results from these experiments
will improve our basic understanding of reproductive development in plants, and will set the basis for a sustained effort
in plant germ line biotechnology, a first step toward a flexible transfer of apomixis into a large variety of sexual crops.
Journal of Plant Biology 05/1998; 41(2):73-81. · 1.07 Impact Factor
