A novel RNA-recognition-motif protein is required for premeiotic G1/S-phase transition in rice (Oryza sativa L.)

The molecular mechanism for meiotic entry remains largely elusive in flowering plants. Only Arabidopsis SWI1/DYAD and maize AM1, both of which are the coiled-coil protein, are known to be required for the initiation of plant meiosis. The mechanism underlying the synchrony of male meiosis, characteristic to flowering plants, has also been unclear in the plant kingdom. In other eukaryotes, RNA-recognition-motif (RRM) proteins are known to play essential roles in germ-cell development and meiosis progression. Rice MEL2 protein discovered in this study shows partial similarity with human proline-rich RRM protein, deleted in Azoospermia-Associated Protein1 (DAZAP1), though MEL2 also possesses ankyrin repeats and a RING finger motif. Expression analyses of several cell-cycle markers revealed that, in mel2 mutant anthers, most germ cells failed to enter premeiotic S-phase and meiosis, and a part escaped from the defect and underwent meiosis with a significant delay or continued mitotic cycles. Immunofluorescent detection revealed that T7 peptide-tagged MEL2 localized at cytoplasmic perinuclear region of germ cells during premeiotic interphase in transgenic rice plants. This study is the first report of the plant RRM protein, which is required for regulating the premeiotic G1/S-phase transition of male and female germ cells and also establishing synchrony of male meiosis. This study will contribute to elucidation of similarities and diversities in reproduction system between plants and other species.     

Expression of a wheat ADP-glucose pyrophosphorylase gene during development of normal and water-stress-affected anthers

In wheat (Triticum aestivum L.), water deficit during meiosis in the microspore mother cells (MMCs) induces pollen abortion, resulting in the failure of fertilization and a reduction in grain set. In stressed plants, meiosis in MMCs proceeds normally but subsequent pollen development is arrested. Unlike normal pollen grains, which accumulate starch during the late maturation phase, stress-affected anthers contain pollen grains with little or no starch. Stress also alters the normal distribution of starch in the anther wall and connective tissue. To determine how starch biosynthesis is regulated within the developing anthers of stressed plants, we studied the expression of ADP-glucose pyrophosphorylase (AGP), which catalyzes the rate limiting step of starch biosynthesis. Two partial-length cDNAs corresponding to the large subunit of AGP were amplified by RT-PCR from anther RNA, and used as probes to monitor AGP expression in developing anthers of normal and water-stressed plants. These clones, WAL1 and WAL2, had identical deduced amino acid sequences and shared 96% sequence identity at the nucleic acid level. In normal anthers, AGP expression was biphasic, indicating that AGP expression is required for starch biosynthesis both during meiosis and later during pollen maturation. AGP expression in stressed anthers was not affected during the first phase of starch accumulation, but was strongly inhibited during the second phase. We conclude from these results that the reduced starch deposition later in the development of stressed pollen could be the result of a lower expression of AGP. However, this inhibition of AGP expression is unlikely to be the primary cause of male sterility because anatomical symptoms of pollen abortion are observed prior to the time when AGP expression is inhibited.     

Pathways to meiotic recombination in Arabidopsis thaliana

Meiosis is a central feature of sexual reproduction. Studies in plants have made and continue to make an important contribution to fundamental research aimed at the understanding of this complex process. Moreover, homologous recombination during meiosis provides the basis for plant breeders to create new varieties of crops. The increasing global demand for food, combined with the challenges from climate change, will require sustained efforts in crop improvement. An understanding of the factors that control meiotic recombination has the potential to make an important contribution to this challenge by providing the breeder with the means to make fuller use of the genetic variability that is available within crop species. Cytogenetic studies in plants have provided considerable insights into chromosome organization and behaviour during meiosis. More recently, studies, predominantly in Arabidopsis thaliana, are providing important insights into the genes and proteins that are required for crossover formation during plant meiosis. As a result, substantial progress in the understanding of the molecular mechanisms that underpin meiosis in plants has begun to emerge. This article summarizes current progress in the understanding of meiotic recombination and its control in Arabidopsis. We also assess the relationship between meiotic recombination in Arabidopsis and other eukaryotes, highlighting areas of close similarity and apparent differences.     

Have a break: determinants of meiotic DNA double strand break (DSB) formation and processing in plants

Meiosis is an essential process for sexually reproducing organisms, leading to the formation of specialized generative cells. This review intends to highlight current knowledge of early events during meiosis derived from various model organisms, including plants. It will particularly focus on cis- and trans-requirements of meiotic DNA double strand break (DSB) formation, a hallmark event during meiosis and a prerequisite for recombination of genetic traits. Proteins involved in DSB formation in different organisms, emphasizing the known factors from plants, will be introduced and their functions outlined. Recent technical advances in DSB detection and meiotic recombination analysis will be reviewed, as these new tools now allow analysis of early meiotic recombination in plants with incredible accuracy. To anticipate future directions in plant meiosis research, unpublished results will be included wherever possible.     

The dsy10 Mutation of Arabidopsis results in desynapsis and a general breakdown in meiosis

The proper pairing and recombination of chromosomes during prophase is essential for the formation of gametes during meiosis. As part of studies to identify genes required for homologous chromosome pairing and recombination during meiosis in plants, we characterized a number of T-DNA-tagged, male-sterile mutants of Arabidopsis. Preliminary cytological studies on one line, 7219 which is male and female sterile, suggested that the mutation may disrupt meiosis and result in the formation of aberrant microsporocytes and microspores. In this report we present the results of a detailed analysis of meiosis in microsporocytes of sterile plants to elucidate the nature of the 7219 mutation. Analysis indicates that the mutation usually results in a desynaptic phenotype, with ten sister chromatids observed prior to metaphase I in most cells. Based on this, we named the mutation dsy10. The presence of several other meiotic defects suggests that dsy10 may not be a typical desynaptic mutant. Received: 15 December 2000 / Accepted: 19 April 2001     

Characterization of Brca2-deficient plants excludes the role of NHEJ and SSA in the meiotic chromosomal defect phenotype

In somatic cells, three major pathways are involved in the repair of DNA double-strand breaks (DBS): Non-Homologous End Joining (NHEJ), Single-Strand Annealing (SSA) and Homologous Recombination (HR). In somatic and meiotic HR, DNA DSB are 5' to 3' resected, producing long 3' single-stranded DNA extensions. Brca2 is essential to load the Rad51 recombinase onto these 3' overhangs. The resulting nucleofilament can thus invade a homologous DNA sequence to copy and restore the original genetic information. In Arabidopsis, the inactivation of Brca2 specifically during meiosis by an RNAi approach results in aberrant chromosome aggregates, chromosomal fragmentation and missegregation leading to a sterility phenotype. We had previously suggested that such chromosomal behaviour could be due to NHEJ. In this study, we show that knock-out plants affected in both BRCA2 genes show the same meiotic phenotype as the RNAi-inactivated plants. Moreover, it is demonstrated that during meiosis, neither NHEJ nor SSA compensate for HR deficiency in BRCA2-inactivated plants. The role of the plant-specific DNA Ligase6 is also excluded. The possible mechanism(s) involved in the formation of these aberrant chromosomal bridges in the absence of HR during meiosis are discussed.     

Expression of female sterility in alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.)

Female sterility associated with the presence of callose in the nucellus at anthesis was studied in an F₁ progeny of two alfalfa plants displaying 5 and 81% ovule sterility. Transgressive segregation was observed and 100% sterile plants were obtained. Two of the sterile plants were used for cytological analyses on sectioned and stain-cleared whole ovules, in comparison to a 100% fertile full sib plant. The first sign of sterility was callose deposition in the nucellus cell walls surrounding the sporogenous cells of the young ovules. At the same stage, no trace of callose was present in ovule primordia of the fertile plant. Megaspore mother cells differentiated in both fertile and sterile ovules and meiosis was initiated, as indicated by chromatin patterning typical of a zygotene stage. However, meiosis was never completed in the sterile plants. In the control, callose was deposited around the meiocyte and as sects between the cells of the dyads and tetrads during meiosis, and disappeared after the completion of meiosis; an embryo sac developed and female fertility was normal. In the sterile ovules, some nucellus cells enlarged and callose accumulation continued forming thick deposits. At anthesis, the sterile ovules lacked an embryo sac and showed massive callose accumulation in the nucellus. Male fertility was normal in female-sterile plants, thus a female-specific arrest of sporogenesis appears to be the cause of sterility. Pistil development was aberrant in some sterile genotypes, even with arrested pistil growth in early flower buds.     

Features of development and reproduction of transgenic flax

Primary transformants carrying a genetic construct with the chimeric gfp-tua6 gene were obtained using biolistic transformation of hypocotyl explants of flax variety Vasilek. Viable modified plants were used as a basis for the production of inbred lines with confirmed inheritance of introduced genetic construct in three generations. The characteristics of phenological growth stages, plant height, number of bolls and meiosis were studied for transgenic plants. A comparison of transformed lines based on reproduction years revealed a significant decrease of seed production in one line. Meiotic analysis of this line at metaphase I and anaphase I stages was conducted. The percentage of cells with impaired meiosis was highest in transgenic plants of the line with the lowest seed production. Thus, the nonspecific incorporation of genetic construct into the flax genome using biolistic transformation impairs meiosis to a different extent and it is the main reason for unequal reproducibility of transgenic flax. The production of stably reproducing transgenic lines requires systematic analysis of meiosis.     

Similarity of domain organization of proteins in phylogenetically distant organisms as a basis of meiosis conservatism

The cytological mechanism of meiosis is very conservative in all eukaryotes. Some meiosis-specific structural proteins of yeasts, nematode Caenorhabditis elegans, Drosophila, and mammals, which play identical roles in cells during meiosis, do not have homology of the primary structure, but their domain organization and conformation are similar. The enzymes of meiotic recombination in yeasts and plants have similar epitopes. These facts suggest that the similarity of the higher level of organization of the meiosis-specific proteins allows these proteins to form similar subcellular structures and produce similar cytological picture of meiosis and similar functions of these subcellular structures. Finally, this leads to a conservative scheme of meiosis in evolutionally distant eukaryotes.     

The chromosomal basis of meiotic acentrosomal spindle assembly and function in oocytes

Several aspects of meiosis are impacted by the absence of centrosomes in oocytes. Here, we review four aspects of meiosis I that are significantly affected by the absence of centrosomes in oocyte spindles. One, microtubules tend to assemble around the chromosomes. Two, the organization of these microtubules into a bipolar spindle is directed by the chromosomes. Three, chromosome bi-orientation and attachment to microtubules from the correct pole require modification of the mechanisms used in mitotic cells. Four, chromosome movement to the poles at anaphase cannot rely on polar anchoring of spindle microtubules by centrosomes. Overall, the chromosomes are more active participants during acentrosomal spindle assembly in oocytes, compared to mitotic and male meiotic divisions where centrosomes are present. The chromosomes are endowed with information that can direct the meiotic divisions and dictate their own behavior in oocytes. Processes beyond those known from mitosis appear to be required for their bi-orientation at meiosis I. As mitosis occurs without centrosomes in many systems other than oocytes, including all plants, the concepts discussed here may not be limited to oocytes. The study of meiosis in oocytes has revealed mechanisms that are operating in mitosis and will probably continue to do so.     

Minichromosome analysis of chromosome pairing, disjunction, and sister chromatid cohesion in maize

With the advent of engineered minichromosome technology in plants, an understanding of the properties of small chromosomes is desirable. Twenty-two minichromosomes of related origin but varying in size are described that provide a unique resource to study such behavior. Fourteen minichromosomes from this set could pair with each other in meiotic prophase at frequencies between 25 and 100%, but for the smaller chromosomes, the sister chromatids precociously separated in anaphase I. The other eight minichromosomes did not pair with themselves, and the sister chromatids divided equationally at meiosis I. In plants containing one minichromosome, the sister chromatids also separated at meiosis I. In anaphase II, the minichromosomes progressed to one pole or the other. The maize (Zea mays) Shugoshin protein, which has been hypothesized to protect centromere cohesion in meiosis I, is still present at anaphase I on minichromosomes that divide equationally. Also, there were no differences in the level of phosphorylation of Ser-10 of histone H3, a correlate of cohesion, in the minichromosomes in which sister chromatids separated during anaphase I compared with the normal chromosomes. These analyses suggest that meiotic centromeric cohesion is compromised in minichromosomes depending on their size and cannot be maintained by the mechanisms used by normal-sized chromosomes.     

Epigenetic regulation of reproductive development and the emergence of apomixis in angiosperms

Apomictic plants reproduce asexually through seeds by avoiding both meiosis and fertilization. While apomixis is genetically controlled, individual loci contributing to its expression have yet to be identified. Here, we review recent results indicating that RNA-dependent DNA methylation pathways acting during female reproduction are essential for proper reproductive development in plants, and may represent key regulators of the differentiation between apomictic and sexual reproduction.     

Random chromosome segregation without meiotic arrest in both male and female meiocytes of a dmc1 mutant of Arabidopsis.

In yeast, the DMC1 gene is required for interhomolog recombination, which is an essential step for bivalent formation and the correct partition of chromosomes during meiosis I. By using a reverse genetics approach, we were able to identify a T-DNA insertion in AtDMC1, the Arabidopsis homolog of DMC1. Homozygotes for the AtDMC1 insertion failed to express AtDMC1, and their residual fertility was 1.5% that of the wild type. Complete fertility was restored in mutant plants when a wild-type copy of the AtDMC1 gene was reintroduced. Cytogenetical analysis points to a correlation of the sterility phenotype with severely disturbed chromosome behavior during both male and female meiosis. In this study, our data demonstrate that AtDMC1 function is crucial for meiosis in Arabidopsis. However, meiosis can be completed in the Arabidopsis dmc1 mutant, which is not the case for mouse or some yeast mutants.     

Similarity of Domain Organization of Proteins in Phylogenetically Distant Organisms as a Basis of Meiosis Conservatism

The cytological mechanism of meiosis is very conservative in all eukaryotes. Some meiosis-specific structural proteins of yeast, nematode Caenorhabditis elegans, Drosophila, and mammals, which play identical roles in cells during meiosis, do not have homology of the primary structure, but their domain organization and conformation are similar. The enzymes of meiotic recombination in yeast and plants have similar epitopes. These facts suggest that the similarity of the higher level of organization of the meiosis-specific proteins allows these proteins to form similar subcellular structures and produce similar cytological picture of meiosis and similar functions of these subcellular structures. Finally, this leads to a conservative scheme of meiosis in evolutionally distant eukaryotes.