Sylvie Jolivet

Turning meiosis into mitosis.

The mutation of as few as three genes in a sexual plant transforms meiosis into mitosis and results in diploid gametes that are genetically identical to the mother plant. This phenotype resembles apomeiosis, which is a major step in apomixis.

Two Meiotic Crossover Classes Cohabit in Arabidopsis: One Is Dependent on MER3,whereas the Other One Is Not

BACKGROUND Crossovers are essential for the completion of meiosis. Recently, two pathways of crossover formation have been identified on the basis of distinct genetic controls. In one pathway, crossover inhibits the occurrence of another such event in a distance-dependent manner. This phenomenon is known as interference. The second kind of crossover is insensitive to interference. The two pathways function independently in budding yeast. Only interference-insensitive crossovers occur in Schizosaccharomyces pombe. In contrast, only interference-sensitive crossovers occur in Caenorabditis elegans. The situation in mammals and plants remains unclear. Mer3 is one of the genes shown to be required for the formation of interference-sensitive crossovers in Saccharomyces cerevisiae. RESULTS To unravel the crossover status in the plant Arabidopsis thaliana, we investigated the role of the A. thaliana MER3 gene through the characterization of a series of allelic mutants. All mer3 mutants showed low levels of fertility and a significant decrease (about 75%) but not a total disappearance of meiotic crossovers, with the number of recombination events initiated in the mutants being similar to that in the wild-type. Genetic analyses showed that the residual crossovers in mer3 mutants did not display interference in one set of adjacent intervals. CONCLUSIONS Mutation in MER3 in Arabidopsis appeared to be specific to recombination events resulting in interference-sensitive crossovers. Thus, MER3 function is conserved from yeast to plants and may exist in other metazoans. Arabidopsis therefore has at least two pathways for crossover formation, one giving rise to interference-sensitive crossover and the other to independently distributed crossovers.

Mutations in AtPS1 (Arabidopsis thaliana Parallel Spindle 1) Lead to the Production of Diploid Pollen Grains

Polyploidy has had a considerable impact on the evolution of many eukaryotes, especially angiosperms. Indeed, most—if not all—angiosperms have experienced at least one round of polyploidy during the course of their evolution, and many important crop plants are current polyploids. The occurrence of 2n gametes (diplogametes) in diploid populations is widely recognised as the major source of polyploid formation. However, limited information is available on the genetic control of diplogamete production. Here, we describe the isolation and characterisation of the first gene, AtPS1 (Arabidopsis thaliana Parallel Spindle 1), implicated in the formation of a high frequency of diplogametes in plants. Atps1 mutants produce diploid male spores, diploid pollen grains, and spontaneous triploid plants in the next generation. Female meiosis is not affected in the mutant. We demonstrated that abnormal spindle orientation at male meiosis II leads to diplogamete formation. Most of the parents heterozygosity is therefore conserved in the Atps1 diploid gametes, which is a key issue for plant breeding. The AtPS1 protein is conserved throughout the plant kingdom and carries domains suggestive of a regulatory function. The isolation of a gene involved in diplogamete production opens the way for new strategies in plant breeding programmes and progress in evolutionary studies.

Synthetic Clonal Reproduction Through Seeds

Clonal reproduction is engineered in a sexual plant by manipulating conserved genes controlling meiosis. Cloning through seeds has potential revolutionary applications in agriculture, because it would allow vigorous hybrids to be propagated indefinitely. However, asexual seed formation or apomixis, avoiding meiosis and fertilization, is not found in the major food crops. To develop de novo synthesis of apomixis, we crossed Arabidopsis MiMe and dyad mutants that produce diploid clonal gametes to a strain whose chromosomes are engineered to be eliminated after fertilization. Up to 34% of the progeny were clones of their parent, demonstrating the conversion of clonal female or male gametes into seeds. We also show that first-generation cloned plants can be cloned again. Clonal reproduction through seeds can therefore be achieved in a sexual plant by manipulating two to four conserved genes.

The Arabidopsis thaliana MND1 homologue plays a key role in meiotic homologous pairing, synapsis and recombination

Mnd1 has recently been identified in yeast as a key player in meiotic recombination. Here we describe the identification and functional characterisation of the Arabidopsis homologue, AtMND1, which is essential for male and female meiosis and thus for plant fertility. Although axial elements are formed normally, sister chromatid cohesion is established and recombination initiation appears to be unaffected in mutant plants, chromosomes do not synapse. During meiotic progression, a mass of entangled chromosomes, interconnected by chromatin bridges, and severe chromosome fragmentation are observed. These defects depend on the presence of SPO11-1, a protein that initiates recombination by catalysing DNA double-strand break (DSB) formation. Furthermore, we demonstrate that the AtMND1 protein interacts with AHP2, the Arabidopsis protein closely related to budding yeast Hop2. These data demonstrate that AtMND1 plays a key role in homologous synapsis and in DSB repair during meiotic recombination.

The endosperm and the embryo of Arabidopsis thaliana are independently transformed through infiltration by Agrobacterium tumefaciens.

Several experiments had indicated that in planta transformation of Arabidopsis thaliana by Agrobacterium involves the female germ line. In order to identify the precise stage at which transformation occurs we have monitored expression of a gusA reporter gene in the two products of the double fertilization of infiltrated plants. The plantlets and the remaining endosperm of seeds were separately tested after germination. It appeared that in the majority of cases only the plantlet or the endosperm were transformed. Based on transformation with two vectors borne by two different Agrobacterium strains, the minority of ‘co-transformed’ plantlets and endosperm can be explained by simultaneous but independent transformation events. These results indicate that mature female gametes could be the targets of T-DNA.

SHOC1, an XPF endonuclease-related protein, is essential for the formation of class I meiotic crossovers.

Crossovers (COs) are essential for the completion of meiosis in most species and lead to new allelic combinations in gametes. Two pathways of meiotic crossover formation have been distinguished. Class I COs, which are the major class of CO in budding yeast, mammals, Caenorhabditis elegans, and Arabidopsis, depend on a group of proteins called ZMM and rely on specific DNA structure intermediates that are processed to form COs. We identified a novel gene, SHOC1, involved in meiosis in Arabidopsis. Shoc1 mutants showed a striking reduction in the number of COs produced, a similar phenotype to the previously described Arabidopsis zmm mutants. The early steps of recombination, revealed by DMC1 foci, and completion of synapsis are not affected in shoc1 mutants. Double mutant analysis showed that SHOC1 acts in the same pathway as AtMSH5, a conserved member of the ZMM group. SHOC1 is thus a novel gene required for class I CO formation in Arabidopsis. Sequence similarity studies detected putative SHOC1 homologs in a large range of eukaryotes including human. SHOC1 appears to be related to the XPF endonuclease protein family, which suggests that it is directly involved in the maturation of DNA intermediates that lead to COs.

Non conservation of the meiotic function of the Ski8/Rec103 homolog in Arabidopsis

Meiotic recombination involves the formation and repair of DNA double‐strand breaks (DSB). One of the genes required for DSB formation in the yeast Saccharomyces cerevisiae, Ski8/Rec103, is intriguing because it also plays a role in cytoplasmic RNA metabolism, a function difficult to relate to DSB formation. The meiotic role of Ski8 is conserved in several fungi, but has not been investigated outside this kingdom. We identified the Ski8 homolog in Arabidopsis thaliana and isolated two mutants. We showed that the Arabidopsis Ski8 homolog was required for normal plant development and growth, suggesting a conserved somatic function, but that it was not required for meiotic recombination or progression. The data presented here provide strong evidence that the meiotic role of Ski8 is not conserved in Arabidopsis and sequence analysis suggests that this may also be the case in a range of other species.

Outcrossing as an Explanation of the Apparent Unconventional Genetic Behavior of Arabidopsis thaliana hth Mutants

The reappearance of HTH alleles in the offspring of homozygous Arabidopsis hth mutants is not consistent with classical Mendelian genetics. It has been suggested that stored RNA may be used to restore genetic information. However, Peng et al. reported that hth mutants tend to display outcrossing and suggested that outcrossing might provide an alternative explanation for the apparent genetic instability. We have confirmed and extended these results, corroborating that the apparent non-Mendelian behavior of hth mutants can be explained by their susceptibility to outcrossing.

Turning rice meiosis into mitosis

Introduction of clonal reproduction through seeds (apomixis) in crops has the potential to revolutionize agriculture by allowing self-propagation of any elite variety, in particular F1 hybrids. In the sexual model plant Arabidopsis thaliana synthetic clonal reproduction through seeds can be artificially implemented by (i) combining three mutations to turn meiosis into mitosis (MiMe) and (ii) crossing the obtained clonal gametes with a line expressing modified CENH3 and whose genome is eliminated in the zygote. Here we show that additional combinations of mutations can turn Arabidopsis meiosis into mitosis and that a combination of three mutations in rice (Oryza sativa) efficiently turns meiosis into mitosis, leading to the production of male and female clonal diploid gametes in this major crop. Successful implementation of the MiMe technology in the phylogenetically distant eudicot Arabidopsis and monocot rice opens doors for its application to any flowering plant and paves the way for introducing apomixis in crop species.