M2WISH: An easy and efficient protocol for whole-mount mRNA in situ hybridization that allows 3D cell resolution of gene expression in Arabidopsis thaliana.

Gene expression analysis is essential for understanding the mechanisms involved in plant development. Here, we developed M2WISH, a protocol based on MicroWave treatment for Wholemount mRNA In Situ Hybridization in Arabidopsis. By permeabilizing tissues without damaging cellular organization this protocol results in high and homogeneous hybridization yields that enable systematic analysis of gene expression dynamics. Moreover, when combined with cellular histochemical staining, M2WISH successfully provides a cellular resolution of gene expression. Thus, we demonstrate the robustness of M2WISH with 10 genes on roots, aerial meristems, leaves, and embryos in the seed. We applied M2WISH to study the spatial dynamics of WUSCHEL (WUS) and CLAVATA3 (CLV3) expression during in vitro meristematic conversion of roots into shoot apical meristems. Thus, we showed that shoot apical meristems could arise from two different types of root structures that differed by their CLV3 gene expression patterns. We constructed 3D cellular representations of WUS and CLV3 gene co-expression pattern and stressed the variability inherent to meristem conversion. Thus, this protocol generates a large amount of data on the localization of gene expression, which can be used to model complex systems.

De novo stem cell establishment in meristems requires repression of organ boundary cell fate.

Stem cells play important roles in animal and plant biology, as they sustain morphogenesis and tissue replenishment following aging or injury. In plants, stem cells are embedded in multicellular structures called meristems. The formation of new meristems is essential for the plastic expansion of the highly branched shoot and root systems. In particular, axillary meristems (AMs) that produce lateral shoots arise from the division of boundary domain cells at the leaf base. The CUP-SHAPED COTYLEDON (CUC) genes are major determinants of the boundary domain and are required for AM initiation. However, how AMs get structured and how stem cells become established de novo remain elusive. Here, we show that two NGATHA-LIKE (NGAL) transcription factors, DEVELOPMENT-RELATED PcG TARGET IN THE APEX4 (DPA4)/NGAL3 and SUPPRESSOR OF DA1-1 7 (SOD7)/NGAL2, redundantly repress CUC expression in initiating AMs of Arabidopsis thaliana. Ectopic boundary fate leads to abnormal growth and organization of the AM and prevents de novo stem cell establishment. Floral meristems of the dpa4 sod7 double mutant show a similar delay in de novo stem cell establishment. Altogether, while boundary fate is required for the initiation of AMs, our work reveals how it is later repressed to allow proper meristem establishment and de novo stem cell niche formation.

The Arabidopsis Cdk1/Cdk2 homolog CDKA;1 controls chromosome axis assembly during plant meiosis.

Meiosis is key to sexual reproduction and genetic diversity. Here, we show that the Arabidopsis cyclin-dependent kinase Cdk1/Cdk2 homolog CDKA;1 is an important regulator of meiosis needed for several aspects of meiosis such as chromosome synapsis. We identify the chromosome axis protein ASYNAPTIC 1 (ASY1), the Arabidopsis homolog of Hop1 (homolog pairing 1), essential for synaptonemal complex formation, as a target of CDKA;1. The phosphorylation of ASY1 is required for its recruitment to the chromosome axis via ASYNAPTIC 3 (ASY3), the Arabidopsis reductional division 1 (Red1) homolog, counteracting the disassembly activity of the AAA+ ATPase PACHYTENE CHECKPOINT 2 (PCH2). Furthermore, we have identified the closure motif in ASY1, typical for HORMA domain proteins, and provide evidence that the phosphorylation of ASY1 regulates the putative self-polymerization of ASY1 along the chromosome axis. Hence, the phosphorylation of ASY1 by CDKA;1 appears to be a two-pronged mechanism to initiate chromosome axis formation in meiosis.

Direct conversion of root primordium into shoot meristem relies on timing of stem cell niche development.

To understand how the identity of an organ can be switched, we studied the transformation of lateral root primordia (LRP) into shoot meristems in Arabidopsis root segments. In this system, the cytokinin-induced conversion does not involve the formation of callus-like structures. Detailed analysis showed that the conversion sequence starts with a mitotic pause and is concomitant with the differential expression of regulators of root and shoot development. The conversion requires the presence of apical stem cells, and only LRP at stages VI or VII can be switched. It is engaged as soon as cell divisions resume because their position and orientation differ in the converting organ compared with the undisturbed emerging LRP. By alternating auxin and cytokinin treatments, we showed that the root and shoot organogenetic programs are remarkably plastic, as the status of the same plant stem cell niche can be reversed repeatedly within a set developmental window. Thus, the networks at play in the meristem of a root can morph in the span of a couple of cell division cycles into those of a shoot, and back, through transdifferentiation.

Identification of ASYNAPTIC4, a Component of the Meiotic Chromosome Axis.

During the leptotene stage of prophase I of meiosis, chromatids become organized into a linear looped array via a protein axis that forms along the loop bases. Establishment of the axis is essential for the subsequent synapsis of the homologous chromosome pairs and the progression of recombination to form genetic crossovers. Here, we describe ASYNAPTIC4 (ASY4), a meiotic axis protein in Arabidopsis (Arabidopsis thaliana). ASY4 is a small coiled-coil protein that exhibits limited sequence similarity with the carboxyl-terminal region of the axis protein ASY3. We used enhanced yellow fluorescent protein-tagged ASY4 to show that ASY4 localizes to the chromosome axis throughout prophase I. Bimolecular fluorescence complementation revealed that ASY4 interacts with ASY1 and ASY3, and yeast two-hybrid analysis confirmed a direct interaction between ASY4 and ASY3. Mutants lacking full-length ASY4 exhibited defective axis formation and were unable to complete synapsis. Although the initiation of recombination appeared to be unaffected in the asy4 mutant, the number of crossovers was reduced significantly, and crossovers tended to group in the distal parts of the chromosomes. We conclude that ASY4 is required for normal axis and crossover formation. Furthermore, our data suggest that ASY3/ASY4 are the functional homologs of the mammalian SYCP2/SYCP3 axial components.

AAA-ATPase FIDGETIN-LIKE 1 and Helicase FANCM Antagonize Meiotic Crossovers by Distinct Mechanisms.

Meiotic crossovers (COs) generate genetic diversity and are critical for the correct completion of meiosis in most species. Their occurrence is tightly constrained but the mechanisms underlying this limitation remain poorly understood. Here we identified the conserved AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) as a negative regulator of meiotic CO formation. We show that Arabidopsis FIGL1 limits CO formation genome-wide, that FIGL1 controls dynamics of the two conserved recombinases DMC1 and RAD51 and that FIGL1 hinders the interaction between homologous chromosomes, suggesting that FIGL1 counteracts DMC1/RAD51-mediated inter-homologue strand invasion to limit CO formation. Further, depleting both FIGL1 and the previously identified anti-CO helicase FANCM synergistically increases crossover frequency. Additionally, we showed that the effect of mutating FANCM on recombination is much lower in F1 hybrids contrasting from the phenotype of inbred lines, while figl1 mutation equally increases crossovers in both contexts. This shows that the modes of action of FIGL1 and FANCM are differently affected by genomic contexts. We propose that FIGL1 and FANCM represent two successive barriers to CO formation, one limiting strand invasion, the other disassembling D-loops to promote SDSA, which when both lifted, leads to a large increase of crossovers, without impairing meiotic progression.

Homoeologous Chromosome Sorting and Progression of Meiotic Recombination in Brassica napus: Ploidy Does Matter!

Meiotic recombination is the fundamental process that produces balanced gametes and generates diversity within species. For successful meiosis, crossovers must form between homologous chromosomes. This condition is more difficult to fulfill in allopolyploid species, which have more than two sets of related chromosomes (homoeologs). Here, we investigated the formation, progression, and completion of several key hallmarks of meiosis in Brassica napus (AACC), a young polyphyletic allotetraploid crop species with closely related homoeologous chromosomes. Altogether, our results demonstrate a precocious and efficient sorting of homologous versus homoeologous chromosomes during early prophase I in two representative B. napus accessions that otherwise show a genotypic difference in the progression of homologous recombination. More strikingly, our detailed comparison of meiosis in near isogenic allohaploid and euploid plants showed that the mechanism(s) promoting efficient chromosome sorting in euploids is adjusted to promote crossover formation between homoeologs in allohaploids. This suggests that, in contrast to other polyploid species, chromosome sorting is context dependent in B. napus.

Crossover localisation is regulated by the neddylation posttranslational regulatory pathway.

Crossovers (COs) are at the origin of genetic variability, occurring across successive generations, and they are also essential for the correct segregation of chromosomes during meiosis. Their number and position are precisely controlled, however the mechanisms underlying these controls are poorly understood. Neddylation/rubylation is a regulatory pathway of posttranslational protein modification that is required for numerous cellular processes in eukaryotes, but has not yet been linked to homologous recombination. In a screen for meiotic recombination-defective mutants, we identified several axr1 alleles, disrupting the gene encoding the E1 enzyme of the neddylation complex in Arabidopsis. Using genetic and cytological approaches we found that axr1 mutants are characterised by a shortage in bivalent formation correlated with strong synapsis defects. We determined that the bivalent shortage in axr1 is not due to a general decrease in CO formation but rather due to a mislocalisation of class I COs. In axr1, as in wild type, COs are still under the control of the ZMM group of proteins. However, in contrast to wild type, they tend to cluster together and no longer follow the obligatory CO rule. Lastly, we showed that this deregulation of CO localisation is likely to be mediated by the activity of a cullin 4 RING ligase, known to be involved in DNA damage sensing during somatic DNA repair and mouse spermatogenesis. In conclusion, we provide evidence that the neddylation/rubylation pathway of protein modification is a key regulator of meiotic recombination. We propose that rather than regulating the number of recombination events, this pathway regulates their localisation, through the activation of cullin 4 RING ligase complexes. Possible targets for these ligases are discussed.

The kinesin AtPSS1 promotes synapsis and is required for proper crossover distribution in meiosis.

Meiotic crossovers (COs) shape genetic diversity by mixing homologous chromosomes at each generation. CO distribution is a highly regulated process. CO assurance forces the occurrence of at least one obligatory CO per chromosome pair, CO homeostasis smoothes out the number of COs when faced with variation in precursor number and CO interference keeps multiple COs away from each other along a chromosome. In several organisms, it has been shown that cytoskeleton forces are transduced to the meiotic nucleus via KASH- and SUN-domain proteins, to promote chromosome synapsis and recombination. Here we show that the Arabidopsis kinesin AtPSS1 plays a major role in chromosome synapsis and regulation of CO distribution. In Atpss1 meiotic cells, chromosome axes and DNA double strand breaks (DSBs) appear to form normally but only a variable portion of the genome synapses and is competent for CO formation. Some chromosomes fail to form the obligatory CO, while there is an increased CO density in competent regions. However, the total number of COs per cell is unaffected. We further show that the kinesin motor domain of AtPSS1 is required for its meiotic function, and that AtPSS1 interacts directly with WIP1 and WIP2, two KASH-domain proteins. Finally, meiocytes missing AtPSS1 and/or SUN proteins show similar meiotic defects suggesting that AtPSS1 and SUNs act in the same pathway. This suggests that forces produced by the AtPSS1 kinesin and transduced by WIPs/SUNs, are required to authorize complete synapsis and regulate maturation of recombination intermediates into COs. We suggest that a form of homeostasis applies, which maintains the total number of COs per cell even if only a part of the genome is competent for CO formation.

Sufficient amounts of functional HOP2/MND1 complex promote interhomolog DNA repair but are dispensable for intersister DNA repair during meiosis in Arabidopsis.

During meiosis, homologous recombination (HR) is essential to repair programmed DNA double-strand breaks (DSBs), and a dedicated protein machinery ensures that the homologous chromosome is favored over the nearby sister chromatid as a repair template. The homologous-pairing protein2/meiotic nuclear division protein1 (HOP2/MND1) protein complex has been identified as a crucial factor of meiotic HR in Arabidopsis thaliana, since loss of either MND1 or HOP2 results in failure of DNA repair. We isolated two mutant alleles of HOP2 (hop2-2 and hop2-3) that retained the capacity to repair meiotic DSBs via the sister chromatid but failed to use the homologous chromosome. We show that in these alleles, the recombinases radiation sensitive51 (RAD51) and disrupted meiotic cDNA1 (DMC1) are loaded, but only the intersister DNA repair pathway is activated. The hop2-2 phenotype is correlated with a decrease in HOP2/MND1 complex abundance. In hop2-3, a truncated HOP2 protein is produced that retains its ability to bind to DMC1 and DNA but forms less stable complexes with MND1 and fails to efficiently stimulate DMC1-driven D-loop formation. Genetic analyses demonstrated that in the absence of DMC1, HOP2/MND1 is dispensable for RAD51-mediated intersister DNA repair, while in the presence of DMC1, a minimal amount of functional HOP2/MND1 is essential to drive intersister DNA repair.

RAD51B plays an essential role during somatic and meiotic recombination in Physcomitrella.

The eukaryotic RecA homologue Rad51 is a key factor in homologous recombination and recombinational repair. Rad51-like proteins have been identified in yeast (Rad55, Rad57 and Dmc1), plants and vertebrates (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1). RAD51 and DMC1 are the strand-exchange proteins forming a nucleofilament for strand invasion, however, the function of the paralogues in the process of homologous recombination is less clear. In yeast the two Rad51 paralogues, Rad55 and Rad57, have been shown to be involved in somatic and meiotic HR and they are essential to the formation of the Rad51/DNA nucleofilament counterbalancing the anti-recombinase activity of the SRS2 helicase. Here, we examined the role of RAD51B in the model bryophyte Physcomitrella patens. Mutant analysis shows that RAD51B is essential for the maintenance of genome integrity, for resistance to DNA damaging agents and for gene targeting. Furthermore, we set up methods to investigate meiosis in Physcomitrella and we demonstrate that the RAD51B protein is essential for meiotic homologous recombination. Finally, we show that all these functions are independent of the SRS2 anti-recombinase protein, which is in striking contrast to what is found in budding yeast where the RAD51 paralogues are fully dependent on the SRS2 anti-recombinase function.

MCM8 is required for a pathway of meiotic double-strand break repair independent of DMC1 in Arabidopsis thaliana.

Mini-chromosome maintenance (MCM) 2-9 proteins are related helicases. The first six, MCM2-7, are essential for DNA replication in all eukaryotes. In contrast, MCM8 is not always conserved in eukaryotes but is present in Arabidopsis thaliana. MCM8 is required for 95% of meiotic crossovers (COs) in Drosophila and is essential for meiosis completion in mouse, prompting us to study this gene in Arabidopsis meiosis. Three allelic Atmcm8 mutants showed a limited level of chromosome fragmentation at meiosis. This defect was dependent on programmed meiotic double-strand break (DSB) formation, revealing a role for AtMCM8 in meiotic DSB repair. In contrast, CO formation was not affected, as shown both genetically and cytologically. The Atmcm8 DSB repair defect was greatly amplified in the absence of the DMC1 recombinase or in mutants affected in DMC1 dynamics (sds, asy1). The Atmcm8 fragmentation defect was also amplified in plants heterozygous for a mutation in either recombinase, DMC1 or RAD51. Finally, in the context of absence of homologous chromosomes (i.e. haploid), mutation of AtMCM8 also provoked a low level of chromosome fragmentation. This fragmentation was amplified by the absence of DMC1 showing that both MCM8 and DMC1 can promote repair on the sister chromatid in Arabidopsis haploids. Altogether, this establishes a role for AtMCM8 in meiotic DSB repair, in parallel to DMC1. We propose that MCM8 is involved with RAD51 in a backup pathway that repairs meiotic DSB without giving CO when the major pathway, which relies on DMC1, fails.

The Arabidopsis HEI10 is a new ZMM protein related to Zip3.

In numerous species, the formation of meiotic crossovers is largely under the control of a group of proteins known as ZMM. Here, we identified a new ZMM protein, HEI10, a RING finger-containing protein that is well conserved among species. We show that HEI10 is structurally and functionally related to the yeast Zip3 ZMM and that it is absolutely required for class I crossover (CO) formation in Arabidopsis thaliana. Furthermore, we show that it is present as numerous foci on the chromosome axes and the synaptonemal complex central element until pachytene. Then, from pachytene to diakinesis, HEI10 is retained at a limited number of sites that correspond to class I COs, where it co-localises with MLH1. Assuming that HEI10 early staining represents an early selection of recombination intermediates to be channelled into the ZMM pathway, HEI10 would therefore draw a continuity between early chosen recombination intermediates and final class I COs.

Epigenetic remodeling of meiotic crossover frequency in Arabidopsis thaliana DNA methyltransferase mutants.

Meiosis is a specialized eukaryotic cell division that generates haploid gametes required for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover (CO). Meiotic CO frequency varies along the physical length of chromosomes and is determined by hierarchical mechanisms, including epigenetic organization, for example methylation of the DNA and histones. Here we investigate the role of DNA methylation in determining patterns of CO frequency along Arabidopsis thaliana chromosomes. In A. thaliana the pericentromeric regions are repetitive, densely DNA methylated, and suppressed for both RNA polymerase-II transcription and CO frequency. DNA hypomethylated methyltransferase1 (met1) mutants show transcriptional reactivation of repetitive sequences in the pericentromeres, which we demonstrate is coupled to extensive remodeling of CO frequency. We observe elevated centromere-proximal COs in met1, coincident with pericentromeric decreases and distal increases. Importantly, total numbers of CO events are similar between wild type and met1, suggesting a role for interference and homeostasis in CO remodeling. To understand recombination distributions at a finer scale we generated CO frequency maps close to the telomere of chromosome 3 in wild type and demonstrate an elevated recombination topology in met1. Using a pollen-typing strategy we have identified an intergenic nucleosome-free CO hotspot 3a, and we demonstrate that it undergoes increased recombination activity in met1. We hypothesize that modulation of 3a activity is caused by CO remodeling driven by elevated centromeric COs. These data demonstrate how regional epigenetic organization can pattern recombination frequency along eukaryotic chromosomes.

Crossovers get a boost in Brassica allotriploid and allotetraploid hybrids.

Meiotic crossovers are necessary to generate balanced gametes and to increase genetic diversity. Even if crossover number is usually constrained, recent results suggest that manipulating karyotype composition could be a new way to increase crossover frequency in plants. In this study, we explored this hypothesis by analyzing the extent of crossover variation in a set of related diploid AA, allotriploid AAC, and allotetraploid AACC Brassica hybrids. We first used cytogenetic methods to describe the meiotic behavior of the different hybrids. We then combined a cytogenetic estimation of class I crossovers in the entire genome by immunolocalization of a key protein, MutL Homolog1, which forms distinct foci on meiotic chromosomes, with genetic analyses to specifically compare crossover rates between one pair of chromosomes in the different hybrids. Our results showed that the number of crossovers in the allotriploid AAC hybrid was higher than in the diploid AA hybrid. Accordingly, the allotetraploid AACC hybrid showed an intermediate behavior. We demonstrated that this increase was related to hybrid karyotype composition (diploid versus allotriploid versus allotetraploid) and that interference was maintained in the AAC hybrids. These results could provide another efficient way to manipulate recombination in traditional breeding and genetic studies.

A high throughput genetic screen identifies new early meiotic recombination functions in Arabidopsis thaliana.

Meiotic recombination is initiated by the formation of numerous DNA double-strand breaks (DSBs) catalysed by the widely conserved Spo11 protein. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation; however, unlike Spo11, few of these are conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we took advantage of a high-throughput meiotic mutant screen carried out in the model plant Arabidopsis thaliana. A collection of 55,000 mutant lines was screened, and spo11-like mutations, characterised by a drastic decrease in chiasma formation at metaphase I associated with an absence of synapsis at prophase, were selected. This screen led to the identification of two populations of mutants classified according to their recombination defects: mutants that repair meiotic DSBs using the sister chromatid such as Atdmc1 or mutants that are unable to make DSBs like Atspo11-1. We found that in Arabidopsis thaliana at least four proteins are necessary for driving meiotic DSB repair via the homologous chromosomes. These include the previously characterised DMC1 and the Hop1-related ASY1 proteins, but also the meiotic specific cyclin SDS as well as the Hop2 Arabidopsis homologue AHP2. Analysing the mutants defective in DSB formation, we identified the previously characterised AtSPO11-1, AtSPO11-2, and AtPRD1 as well as two new genes, AtPRD2 and AtPRD3. Our data thus increase the number of proteins necessary for DSB formation in Arabidopsis thaliana to five. Unlike SPO11 and (to a minor extent) PRD1, these two new proteins are poorly conserved among species, suggesting that the DSB formation mechanism, but not its regulation, is conserved among eukaryotes.

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.

The Arabidopsis BLAP75/Rmi1 homologue plays crucial roles in meiotic double-strand break repair.

In human cells and in Saccharomyces cerevisiae, BLAP75/Rmi1 acts together with BLM/Sgs1 and TopoIIIalpha/Top3 to maintain genome stability by limiting crossover (CO) formation in favour of NCO events, probably through the dissolution of double Holliday junction intermediates (dHJ). So far, very limited data is available on the involvement of these complexes in meiotic DNA repair. In this paper, we present the first meiotic study of a member of the BLAP75 family through characterisation of the Arabidopsis thaliana homologue. In A. thaliana blap75 mutants, meiotic recombination is initiated, and recombination progresses until the formation of bivalent-like structures, even in the absence of ZMM proteins. However, chromosome fragmentation can be detected as soon as metaphase I and is drastic at anaphase I, while no second meiotic division is observed. Using genetic and imunolocalisation studies, we showed that these defects reflect a role of A. thaliana BLAP75 in meiotic double-strand break (DSB) repair -- that it acts after the invasion step mediated by RAD51 and associated proteins and that it is necessary to repair meiotic DSBs onto sister chromatids as well as onto the homologous chromosome. In conclusion, our results show for the first time that BLAP75/Rmi1 is a key protein of the meiotic homologous recombination machinery. In A. thaliana, we found that this protein is dispensable for homologous chromosome recognition and synapsis but necessary for the repair of meiotic DSBs. Furthermore, in the absence of BLAP75, bivalent formation can happen even in the absence of ZMM proteins, showing that in blap75 mutants, recombination intermediates exist that are stable enough to form bivalent structures, even when ZMM are absent.

Zip4/Spo22 is required for class I CO formation but not for synapsis completion in Arabidopsis thaliana.

In budding yeast meiosis, the formation of class I interference-sensitive crossovers requires the ZMM proteins. These ZMM proteins are essential in forming a mature synaptonemal complex, and a subset of these (Zip2, Zip3, and Zip4) has been proposed to compose the core of synapsis initiation complexes (SICs). Zip4/Spo22 functions with Zip2 to promote polymerization of Zip1 along chromosomes, making it a crucial SIC component. In higher eukaryotes, synapsis and recombination have often been correlated, but it is totally unknown how these two processes are linked. In this study, we present the characterization of a higher eukaryote SIC component homologue: Arabidopsis AtZIP4. We show that mutations in AtZIP4 belong to the same epistasis group as Atmsh4 and eliminate approximately 85% of crossovers (COs). Furthermore, genetic analyses on two adjacent intervals of Chromosome I established that the remaining COs in Atzip4 do not show interference. Lastly, immunolocalization studies showed that polymerization of the central element of the synaptonemal complex is not affected in Atzip4 background, even if it may proceed from fewer sites compared to wild type. These results reveal that Zip4 function in class I CO formation is conserved from budding yeast to Arabidopsis. On the other hand, and contrary to the situation in yeast, mutation in AtZIP4 does not prevent synapsis, showing that both aspects of the Zip4 function (i.e., class I CO maturation and synapsis) can be uncoupled.