ZmHOP2a Coordinates Meiotic Recombination and Synapsis in Maize

Abstract

Accurate meiotic chromosome segregation requires tight coordination between homologous recombination and synaptonemal complex (SC) assembly. HOP2 is a conserved recombination factor that acts with MND1 to promote homolog pairing, but whether it also contributes to SC assembly in plants remains unclear. Here, we show that the maize (Zea mays L.) HOP2 homolog, ZmHOP2a, is required for both recombination progression and synapsis. In zmhop2a mutants, meiotic double-strand break formation was largely unaffected, as indicated by normal γH2AX accumulation. Nevertheless, RAD51 loading, homologous pairing, and synapsis were partially impaired. Cytological analyses further revealed that the axis-associated proteins AFD1 and ASY1 localized normally in mutant meiocytes, but assembly of the transverse filament protein ZYP1 was defective. ZmHOP2a displayed a dynamic spatiotemporal localization pattern during prophase Ⅰ and transiently associated with chromosomes during ZYP1 loading. Together, our findings suggest that ZmHOP2a functions at the interface between recombination progression and SC assembly to promote proper synapsis in maize meiosis.

Keywords

meiosis / ZmHOP2a / SC assembly

1 Introduction

Meiosis is an evolutionarily conserved process that enables the production of haploid gametes from diploid precursors through one round of DNA replication followed by two nuclear divisions (de la Fuente et al., 2021). During prophase I, homologous chromosomes pair, undergo recombination, and become closely juxtaposed via the synaptonemal complex (SC), a tripartite proteinaceous structure comprising two parallel axial/lateral elements (AEs/LEs) connected by transverse filaments (TFs) and central-element (CE) proteins (Leu et al., 1998; Sakuno et al., 2022). Although SC architecture is broadly conserved, its protein constituents exhibit limited sequence similarity across species (Higgins et al., 2005; Llano and Pendás, 2023). The chromosome axis, established by proteins such as ASY1, ASY3 (and its maize homolog DSY2), and the meiosis-specific cohesin REC8, provides the structural foundation for double-strand break (DSB) formation and SC assembly (Armstrong et al., 2002; Sanchez-Moran et al., 2008; Wang et al., 2010). Upon axis assembly, TFs polymerize between homologs during synapsis, a process that is stabilized by other CE components. Notably, studies in Arabidopsis thaliana and rice have revealed that CE proteins are not merely structural scaffolds but actively coordinate recombination progression with synapsis (Miao et al., 2013; Vrielynck et al., 2023).

In many organisms, the repair of meiotic DSBs is tightly coupled with homologous pairing and synapsis. Mutants defective in DSB formation or recombination progression often show aberrant pairing and SC assembly, whereas SC components in turn influence the maturation of recombination intermediates into crossovers (COs). For instance, in Arabidopsis, the loss of CE proteins SCEP1 or SCEP2 disrupts synapsis and alters CO interference without altering the CO number (Vrielynck et al., 2023). Similarly, in rice (Oryza sativa), the PCH2 homolog CRC1 localizes to the central region of the SC and is essential for the initiation of recombination and the recruitment of the axis-associated protein OsPAIR2 (Miao et al., 2013). These findings underscore that SC central-region components not only stabilize synapsis but also actively coordinate the recombination machinery. However, relatively few SC proteins have been functionally characterized in maize and other major crops, and the mechanistic links between DSB repair factors and the physical assembly of the SC remain poorly understood.

The formation and repair of meiotic DSBs depend on the conserved recombinases RAD51 and DMC1, assisted by accessory factors that promote homology search and strand invasion. The HOP2–MND1 heterodimer, first identified in Saccharomyces cerevisiae, plays an essential role in stabilizing recombination intermediates and ensuring accurate homolog pairing (Bugreev et al., 2014; Pezza et al., 2014; Tsubouchi, 2023). In mice, inactivation of HOP2 leads to meiotic arrest, defective DSB repair, and aberrant synapsis between nonhomologous chromosomes (de la Fuente et al., 2021). However, whether HOP2 additionally contributes to SC assembly or modulates CO maturation through physical interactions with SC components has not been established in plants.

Here, we identified the maize (Zea mays L.) HOP2 homolog, ZmHOP2a, and isolated two independent mutant alleles to investigate its meiotic function. Our analyses reveal that ZmHOP2a is essential for preventing nonhomologous chromosome associations and for proper SC assembly; consequently, zmhop2a mutants exhibit defective synapsis and reduced CO frequency. Using immunolocalization with a ZmHOP2a-specific antibody, we further demonstrate that ZmHOP2a displays a dynamic spatiotemporal pattern during prophase I, resembling the behavior of an axial-element component. Collectively, these results suggest that ZmHOP2a functions at the interface between DSB repair and SC assembly, potentially linking recombination progression with synapsis in maize.

2 Materials and Methods

2.1 Plant materials

The maize (Zea mays) mutant alleles zmhop2a-1 (EMS4-123591) and zmhop2a-2 (EMS4-279922) in the B73 inbred background were obtained from the Maize EMS-induced Mutant Database (MEMD) (Lu et al., 2018). The zmasy1 mutant was reported previously (Wang et al., 2023). All plants were cultivated and fertilized in the field during the normal growing season or in a controlled environment room. Nicotiana benthamiana (tobacco) plants were grown in the greenhouse under a 16-h light and 8-h dark photoperiod. Maize mesophyll protoplasts were isolated from second leaves of 2-week-old etiolated B73 seedlings.

2.2 cDNA cloning and real-time PCR for transcript expression assay

Total RNA was isolated from SAM, IM, tassel, leaf, root, stem, embryo, endosperm, and anthers of different lengths. First-strand cDNA synthesis was performed using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, 6210A) with the oligo(dT) primer. Quantitative real-time PCR was performed with SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The maize Ubiquitin gene was used as an internal control.

2.3 Observation of pollen viability

Pollen viability was evaluated by Alexander staining as previously described (Johnson-Brousseau and McCormick, 2004). Anthers were collected from wild-type and zmhop2a mutant plants at anthesis. Pollen grains were released from the anthers into Alexander staining solution and examined under a Leica EZ4 HD stereomicroscope. Images of stained pollen grains were acquired using a Leica DM2000 LED microscope.

2.4 Meiotic chromosome preparation

Chromosome spreads were prepared from young tassels fixed in Carnoy’s solution (ethanol:acetic acid, 3:1 [v/v]). After fixation for 30 min at room temperature, the samples were stored in 75% ethanol at 4°C until examined. The samples were squashed in a drop of 45% acetic acid. The slides were frozen in liquid nitrogen, and the coverslips were removed immediately. After dehydration in an ethanol gradient series (70%, 90%, and 100%) for 5 min each, the slides were air dried. Chromosomes were stained with 10 μL DAPI in antifade mounting medium (VECTASHIELD, Vector Laboratories). Images were acquired using a Nikon Ci-S-FL fluorescence microscope equipped with a DS-Qi2 microscope camera system.

2.5 Fluorescence in situ hybridization

Fluorescently labeled dUTP nucleotides (Roche, Basel, Switzerland) were incorporated into the probes by nick translation as previously described. DIG-labeled probes were detected using an anti-digoxigenin antibody (Vector Laboratories). The pAtT4 clone contains telomeric repeats, and the pTa794 clone contains 5S rDNA sequences (Li and Arumuganathan, 2001). Chromosomes were counterstained with 10 μL DAPI in antifade mounting medium (VECTASHIELD, Vector Laboratories). Chromosome images were captured using a Nikon Ci-S-FL fluorescence microscope equipped with a DS-Qi2 microscope camera (Nikon, Tokyo, Japan).

2.6 Antibody production

To generate antibodies against HOP2a, ASY1, and the C-terminal region of ZYP1, the corresponding cDNA fragments were cloned into the expression vector pET-28a (TransGen). The resulting constructs were transformed into the E. coli expression strain Transetta (TransGen). Recombinant protein expression was induced at 16 °C for 7 h, and protein purification was carried out as described previously (Jing et al., 2019). Polyclonal antibodies against HOP2a, ASY1, and ZYP1-Cter were raised in rabbits, rats, and guinea pigs, respectively, by HUABIO. These antibodies were used at a dilution of 1:100 for immunocytology.

2.7 Immunofluorescence assay and image analysis

For immunofluorescence assays, the young tassels were fixed with fresh 4% (w/v) paraformaldehyde for 30 min. The procedure was performed as described previously (Wang et al., 2006). Images were captured under a Ci-S-FL fluorescence microscope (Nikon) equipped with a DS-Qi2 microscopy camera (Nikon). High-resolution images were captured under an IX83 fluorescence microscope (Olympus) equipped with a STEDYCON microscopy camera (Abberior, Heidelberg, Germany).

2.8 Yeast two-hybrid (Y2H) assay

The yeast two-hybrid (Y2H) assays were conducted using the Matchmaker Yeast Two-Hybrid System (Clontech). The full-length CDS of ZmHOP2a and its truncated fragments (HOP2a, HOP2a-WHD, HOP2a-LZ1, HOP2a-LZ2, HOP2a-LZ3wCH, HOP2a-LZ1-LZ2, and HOP2a-SMC) were independently cloned into the pGBKT7 or pGADT7 vector. The resulting bait and prey constructs were co-transformed into the yeast strain AH109. Co-transformants were first selected on SD/DDO (SD/-Leu/-Trp) medium at 30°C for 3–4 days. Positive interactions were identified on SD/QDO (SD/-Leu/-Trp/-His/-Ade) medium. The procedures were performed according to the manufacturer’s protocol (Clontech).

2.9 Split-luciferase complementation assay

For the split-LUC assay, the full-length CDSs of ZYP1 and ZmHOP2a were inserted into the JW771 (NLuc) and JW772 (CLuc) vectors, respectively. The Agrobacterium tumefaciens strain GV3101 carrying the corresponding vectors, together with empty NLuc, CLuc, and the p19 silencing plasmid, was cultured and mixed for infiltration into the leaves of Nicotiana benthamiana. After 48 h, 1 mM luciferin was infiltrated into the leaves, and LUC signals were detected using the cooled Tanon-4600 imaging system. Three biological replicates were performed.

2.10 Bimolecular fluorescence complementation assay

To conduct BiFC assays, the full-length CDSs of ZYP1 and ZmHOP2a were ligated into BiFC vectors, including pSCYNE (SCN) and pSCYCE (SCC). The constructed plasmids were transformed into maize protoplasts by PEG-mediated transformation as described previously (Bart et al., 2006). After incubation in the dark for 12 h at 37 °C, the CFP signals were captured under a confocal laser scanning microscope at an excitation wavelength of 405 nm (LSM770, Zeiss, Oberkochen, Germany).

2.11 Subcellular localization

To determine the subcellular localization of ZmHOP2a, the full-length CDS without the stop codon was cloned into the pGreen-GFP vector to generate the pUbi::ZmHOP2a-GFP construct. The pUbi::ZmHOP2a-GFP and empty pUbi::GFP vectors were transformed into maize protoplasts and incubated in the dark at 25 °C for 12 h. GFP fluorescence was visualized using the LSM900 confocal microscope (Zeiss, Oberkochen, Germany) with an excitation wavelength of 488 nm.

2.12 Accession numbers

Sequence data used in this article can be found in GenBank/National Center for Biotechnology Information (NCBI) under the following accession numbers: ZmHOP2a: NP_001149081.1; ZmHOP2b: XP_008665283.1. Accession numbers of different HOP2 homologs used in the neighbor-joining tree construction are as follows: Sorghum bicolor: XP_002464006.1; Setaria italica: XP_004982010.1; Oryza sativa: XP_015632741.1; Hordeum vulgare: XP_044979970.1; Arabidopsis thaliana, CAF28783.1; Pisum sativum, XP_050905123; Brassica napus: XP_013698448.1; Phoenix dactylifera: XP_008781157.1; Homo sapiens: NP_057640.1; Mus musculus: NP_032975.1; Saccharomyces cerevisiae: XP_001704033.1.

3 Results

3.1 Characterization of ZmHOP2a

As HOP2 is a highly conserved protein, the reported protein sequence of Arabidopsis AtHOP2 was used as a query in a BLASTP search against the maize genome database (MaizeGDB). Two candidate gene models (Zm00001eb216450 and Zm00001eb054780) were identified with significant similarities. They were designated as ZmHOP2a and ZmHOP2b, respectively. An RT-PCR analysis showed that ZmHOP2a was highly expressed in the shoot apical meristem, inflorescence meristem, and anthers, whereas ZmHOP2b was weakly expressed in these tissues (Fig. 1-A). These results suggest that ZmHOP2a is the major expressed HOP2 homolog in maize.

ZmHOP2a encodes a 227-amino acid protein with a conserved winged-helix domain and three predicted leucine zipper regions (Fig. S1-B and -C). Transient expression of ZmHOP2a-GFP in maize protoplasts showed that the protein was localized in the nucleus (Fig. S2). To investigate the function of ZmHOP2a in maize meiosis, we obtained two ethyl methanesulfonate (EMS)-induced mutants (named zmhop2a-1 and zmhop2a-2) from the Maize EMS-induced Mutant Database (MEMD). The zmhop2a-1 allele in the B73 background was caused by a C-to-A change in the third exon of ZmHOP2a, resulting in a premature stop codon, whereas zmhop2a-2 had a G-to-A substitution at the splice donor site of intron 4, producing a shorter RT-PCR product consistent with altered splicing and predicted exon 5 deletion in the mature mRNA (Fig. 1-B). The predicted ZmHOP2a-1 protein lacks the conserved LZ1 region (Fig. S1-A and -B), an important structural domain required for HOP2 function. The absence of this domain is expected to impair HOP2 activity and may contribute to the loss-of-function phenotype of the mutant. A phylogenetic tree was constructed using ZmHOP2a and its homologs from other species. This tree showed that ZmHOP2a clustered within the monocot HOP2 clade (Fig. S1-C).

Both zmhop2a-1 and zmhop2a-2 plants showed normal vegetative growth and development but were completely male sterile (Fig. 1-C; Fig. S3-A). When pollinated with wild-type pollen, neither mutant produced seeds (Fig. 1-D; Fig. S3-B), indicating that female fertility was also impaired. Alexander staining showed that wild-type pollen grains were spherical and stained dark brown, whereas mutant pollen grains were shrunken and unstained (Fig. 1-E; Fig. S3-C and -D). In the progeny of heterozygous plants, 189 individuals were fertile and 65 were sterile, fitting the expected 3:1 segregation ratio (χ² = 1.3, P > 0.05), implying that sterility in zmhop2a is caused by a monogenic recessive mutation. These results indicate that ZmHOP2a is required for fertility in maize.

3.2 Abnormal chromosome behavior in zmhop2a mutants

To investigate the cause of sterility in zmhop2a mutants, meiotic chromosome behavior was examined by DAPI staining in wild-type (WT) and zmhop2a-1 meiocytes. In WT, homologous chromosomes were fully synapsed at pachytene and appeared as thick threads (Fig. 2-A to -C). At diakinesis, 10 bivalents were observed (Fig. 2-D), which then aligned on the equatorial plate at metaphase I (Fig. 2-E). Homologous chromosomes separated at anaphase I (Fig. 2-F), and after meiosis II, equal chromosome segregation produced normal tetrads (Fig. 2-G) and tetrads (Fig. 2-H). Compared with WT, chromosome behavior in zmhop2a-1 was similar from leptotene to zygotene (Fig. 2-I and -J), but clear abnormalities appeared thereafter. At pachytene, only partial synapsis was observed, and many chromosomes remained as single thin threads, indicating extensive asynapsis (Fig. 2-K). At diakinesis, canonical bivalents were largely absent and most chromosomes appeared as univalents (Fig. 2-L to -N; Fig. S5-B). At anaphase I, chromosomes segregated unequally, frequently producing lagging chromosomes, micronuclei, and chromosome fragments (Fig. 2-O; Fig. S5-F to -H). As a consequence, abnormal dyads and unequal tetrads with micronuclei were formed after meiosis II (Fig. 2-P; Fig. S6).

Chiasma frequency was significantly reduced in zmhop2a-1, with the average chiasma number reduced by 60.15% compared to WT (WT, 16.44 ± 2.46, n = 25; mutant, 6.55 ± 1.73, n = 53; P < 0.0001; Fig. S5), indicating severely impaired meiotic recombination. Similar meiotic defects were observed in zmhop2a-2 (Fig. S4). The comparable phenotypes of the two independent alleles support that the observed defects are associated with disruption of ZmHOP2a function. Therefore, zmhop2a-1 was selected as a representative allele for subsequent detailed analyses. Altogether, our results indicate that defects in chromosome interactions and segregation underlie the sterility of zmhop2a mutants.

3.3 Homologous chromosome pairing is impaired in zmhop2a

Telomere bouquet formation, an evolutionarily conserved chromosome arrangement in eukaryotes, brings telomeres together in a restricted region of the nuclear envelope during early zygotene and is thought to promote the initiation of homologous chromosome pairing (Bass et al., 1997; Harper et al., 2004). To examine whether zmhop2a-1 affects telomere bouquet formation, we conducted a fluorescence in situ hybridization (FISH) analysis in wild-type and zmhop2a-1 meiocytes using pAtT4 as a telomere-specific probe. The FISH results revealed a typical bouquet configuration in both wild-type (n = 12) and zmhop2a-1 (n = 24) meiocytes at zygotene (Fig. 3-A and -B), indicating that bouquet formation was normal in the mutant.

To further determine whether homologous chromosome pairing was defective in zmhop2a-1, we performed FISH analysis using 5S rDNA as a probe, a tandem repetitive sequence located on the long arm of chromosome 2 and commonly used to assess chromosome pairing and segregation in maize. In wild-type pachytene meiocytes, only one 5S rDNA signal was detected (n = 15, Fig. 3-C), indicating that homologous chromosomes were properly paired. In contrast, two distinct 5S rDNA signals were observed in zmhop2a-1 pachytene meiocytes (n = 35, Fig. 3-D). This result indicated that homologous chromosome pairing was impaired in zmhop2a-1.

3.4 ZmHOP2a is indispensable for synaptonemal complex assembly

The synaptonemal complex (SC) is a proteinaceous structure required for homologous chromosome synapsis and CO formation during meiosis (Woglar and Villeneuve, 2018). In maize, the cohesin protein AFD1 and the HORMA-domain protein ASY1 localize along meiotic chromosome axes and mark the axial elements (AEs) during early prophase I (Golubovskaya et al., 2006; Lambing et al., 2015). To determine whether ZmHOP2a was required for SC assembly, we examined the localization of AFD1, ASY1, and ZYP1 in wild-type and zmhop2a-1 meiocytes by immunostaining.

In wild-type meiocytes, AFD1 appeared as continuous linear signals along the full length of chromosomes at zygotene (Fig. 4-A, n = 24). A similar AFD1 loading pattern was observed in zmhop2a-1 meiocytes (Fig. 4-B, n = 10), indicating that ZmHOP2a was not required for AFD1 deposition. Likewise, ASY1 formed extensive linear signals along chromosomes in wild-type zygotene meiocytes (Fig. 4-C, n = 58) and showed a comparable localization pattern in zmhop2a-1 (Fig. 4-D, n = 11), suggesting that AE assembly occurred normally in the mutant.

We next analyzed ZYP1, the transverse filament protein of the SC central region in maize. In wild-type pachytene meiocytes, ZYP1 formed continuous linear stretches along bivalents (Fig. 4-E, n = 34). By contrast, in zmhop2a-1 pachytene meiocytes, ZYP1 signals were detected only as punctate foci and short stretches (Fig. 4-F, n = 10), indicating that central region assembly was severely impaired in the absence of ZmHOP2a. Furthermore, examination of meiocytes from mid- to late prophase I showed that, unlike in the wild type, ASY1 signals in zmhop2a-1 remained at similar intensity on synapsed and unsynapsed chromosomal regions (Fig. S7). This observation suggests that ASY1 removal from chromosome axes was delayed in the mutant. Taken together, these results indicate that although ZmHOP2a is dispensable for AE formation, it is required for proper SC assembly and chromosome axis remodeling during maize meiosis.

3.5 ZmHOP2a transiently associates with chromosome axes during early synapsis

To further define the relationship between ZmHOP2a and chromosome axis components, we performed immunolocalization using an anti-ZmHOP2a antibody in wild-type meiocytes. ZmHOP2a signals were detected throughout prophase I and were consistently observed around the nucleolar periphery (Fig. S8-A to -E). No signal was detected in zmhop2a mutant meiocytes (Fig. S8-F), supporting the specificity of the antibody in immunocytological analyses.

To examine the localization dynamics of ZmHOP2a during meiosis, we carried out co-immunolocalization analyses with ASY1 and ZYP1. ZmHOP2a first appeared as a diffuse nuclear signal at leptotene and remained so through early zygotene (Fig. S8-A; Fig. 5-A). At mid-zygotene, ZmHOP2a formed short chromosome-associated linear signals (Fig. 5-B). By late zygotene, these signals closely paralleled ASY1 along chromosome axes (Fig. 5-C). However, the two proteins showed distinct distributions at earlier zygotene stages. Dual immunolocalization further showed that ZmHOP2a was associated with sites of synapsis and persisted as foci and short linear stretches along synapsing chromosomes from zygotene to pachytene. Colocalization analysis yielded a Pearson’s correlation coefficient of 0.82 for ZmHOP2a and ASY1 (n = 8), indicating a high degree of colocalization between the two proteins (Fig. S9-A). Super-resolution imaging showed that the short linear ZmHOP2a signals had a mean length of 202 nm (range = 140–330 nm; n = 10 per cell; Fig. 5-H, Table S1). In a larger sample analyzed by conventional fluorescence microscopy, an average of 10 such foci per nucleus was observed (n = 60). Intensity profiling across these structures revealed bimodal distributions for both ZmHOP2a and ASY1, with the ZmHOP2a signals lying between two ASY1 peaks (Fig. 5-G).

We next examined the relationship between ZmHOP2a and ZYP1 during SC assembly. As ZYP1 began to load onto chromosome axes, ZmHOP2a was progressively redistributed from axis-associated structures. At sites marked by short nascent ZYP1 stretches, ZmHOP2a was no longer confined to the axes, but instead became more diffuse in the surrounding chromatin, although short linear signals were still observed (Fig. 5-D, n = 5). By early pachytene, ZYP1 formed continuous linear signals along synapsed chromosomes (Fig. 5-E, n = 11), whereas ZmHOP2a became diffusely distributed around the chromosomes. At mid-pachytene, ZYP1 remained continuously associated with chromosome axes, while ZmHOP2a signals became weaker and more sparsely distributed (Fig. 5-F, n = 6).

Consistent with these observations, the spatial correlation between ZmHOP2a and ZYP1 (r = 0.5815, R² = 0.3382) was lower than that between ZmHOP2a and ASY1 (r = 0.7440, R² = 0.5535) (Fig. 5-G and -I; Fig. S9-A; Table S1 to 2). Super-resolution imaging further showed that the short linear ZmHOP2a signals were consistently flanked by ASY1 signals on both sides (Fig. 5-H). After full ZYP1 loading, the mean distance between two ZmHOP2a signal peaks was 153 nm (range = 65–205 nm; Fig. S9-B). The enlarged view showed that ZmHOP2a puncta lay adjacent to, rather than overlapping with, ZYP1-labeled stretches (Fig. 5-J). Together, these data indicate that ZmHOP2a transiently associates with chromosome axes during early synapsis and is progressively redistributed as ZYP1 polymerizes along the SC.

3.6 ZmHOP2a is essential for meiotic DSB repair

Because abnormal chromosome behavior was observed in zmhop2a-1 meiocytes, we asked whether meiotic DSB formation and early stages of DSB repair were affected in the mutant. We therefore performed immunostaining using antibodies against γH2AX and RAD51. γH2AX, the phosphorylated form of H2AX, is widely used as a cytological marker for DSBs (Stucki and Jackson, 2006). In wild-type meiocytes, abundant γH2AX signals were observed at zygotene (Fig. 6-A, n = 46). Similar signals were detected in zmhop2a-1 meiocytes at the same stage (Fig. 6-B, n = 38), and the number of γH2AX foci did not differ significantly between the two genotypes (Fig. 6-C), indicating that meiotic DSB formation is not markedly affected in zmhop2a-1. RAD51 is a marker for early meiotic recombination events (Pawlowski et al., 2003). RAD51 foci were detected in both wild-type and zmhop2a-1 meiocytes at zygotene (Fig. 6-D and -E), and their numbers were moderately reduced in zmhop2a-1 compared with the wild-type (Fig. 6-F), indicating that partial defects in DSB repair upon early RAD51 loading in zmhop2a-1.

In yeast two-hybrid (Y2H) assays, full-length ZmHOP2a interacted with COM1 and RAD50 in yeast cells (Fig. S10). COM1 and RAD50 are known to function in meiotic DSB processing and SPO11 removal. These results suggest that ZmHOP2a may be associated with meiotic DSB repair or processing.

3.7 ZmHOP2a physically interacts with ZYP1 but not ASY1

To test whether ZmHOP2a directly interacts with components of the synaptonemal complex, we performed Y2H assays focusing on the axial element protein ASY1 and the transverse filament protein ZYP1. Although ZmHOP2a and ASY1 showed strong colocalization on meiotic chromosomes, no direct interaction between the two proteins was detected in yeast. Moreover, ZmHOP2a signals at zygotene in zmasy1 meiocytes were comparable to those in wild type (Fig. S11), indicating that ZmASY1 is not required for ZmHOP2a loading. Consequently, the meiotic phenotype of the zmasy1 zmhop2a-1 double mutant resembled that of the zmhop2a-1 single mutant, with no obvious chromosome aggregation or fragmentation (Fig. S12). These data suggest that ZmHOP2a and ASY1 localize independently on meiotic chromosomes.

In contrast, ZmHOP2a interacted with ZYP1 in yeast, and this interaction was further supported by Bimolecular Fluorescence Complementation (BiFC) and Luciferase Complementation Imaging (LCI) assays (Fig. 7). Deletion analysis showed that amino acids 30–68 of ZmHOP2a are sufficient for its interaction with ZYP1, whereas the maize-specific SMC domain is dispensable for this interaction (Fig. 7-A).

4 Discussion

4.1 Functional conservation and divergence of HOP2 in maize meiosis

HOP2 is a conserved meiotic factor in eukaryotes, but its functional contribution varies among species (Tsubouchi, 2023). In plants, HOP2 has been implicated in homologous recombination, including CO formation, and the prevention of illegitimate interactions between nonhomologous chromosomes (Uanschou et al., 2013; Shi et al., 2019; Tafreshi et al., 2022). In yeast and mammals, hop2 mutants arrest at prophase I, whereas Arabidopsis meiosis progresses to completion, indicating divergence in the meiotic requirement for HOP2 among lineages (Leu et al., 1998). Moreover, although HOP2 generally functions together with MND1 (MEIOTIC NUCLEAR DIVISION PROTEIN1), mammalian studies suggest that HOP2 may also have MND1-independent activities (Pezza et al., 2014). Whether ZmHOP2a interacts with ZmMND1 and whether such interaction contributes to the observed defects in recombination and synapsis remain to be investigated.

Our results show that maize ZmHOP2a is the major functional HOP2 homolog during meiosis. Although two HOP2-related paralogs are present in the maize genome, only ZmHOP2a is detectably expressed in reproductive tissues, whereas ZmHOP2b appears transcriptionally inactive. The zmhop2a-1 mutant exhibits male and female sterility, defective homolog pairing, delayed or incomplete synapsis, and impaired DSB repair, indicating that ZmHOP2a plays an important role in multiple steps of meiotic progression. In contrast to rice oshop2 mutants, which predominantly display univalent formation, zmhop2a-1 meiocytes frequently show both univalents and entangled chromosomes. These phenotypic differences may arise from a combination of HOP2 functional divergence, differences in genome organization and complexity. Comparison with Arabidopsis further supports functional diversification of HOP2, as different hop2 alleles in Arabidopsis cause distinct meiotic defects ranging from chromosome fragmentation to unequal segregation (Vignard et al., 2007; Uanschou et al., 2013).

Taken together, these observations suggest that ZmHOP2a plays an important role in homolog pairing, SC assembly, and meiotic recombination in maize. The interaction between ZmHOP2a and ZYP1 further supports a potential role for ZmHOP2a at the interface between chromosome synapsis and recombination. In addition, ZmHOP2a contains a putative SMC-related domain that has not been reported in other plant HOP2 homologs (Chen et al., 2021). Although its biological significance remains to be determined, this feature raises the possibility that ZmHOP2a has acquired a maize-specific role in meiotic chromosome organization.

4.2 ZmHOP2a is related to central element components

Recent studies have redefined the synaptonemal complex (SC) as a highly dynamic proteinaceous machine rather than a static, ladder-like structure (Zhao et al., 2024). In yeast and worms, the central region of the SC exhibits fluid-like properties, permitting weakly bound proteins to diffuse within the structure (Yang et al., 2023). During maize prophase I, ZmHOP2a undergoes dynamic repositioning from a diffuse perinuclear pattern at leptotene to short, linear foci partially overlapping the axial element protein ASY1 at early pachytene, and becomes largely excluded from regions fully loaded with the transverse filament protein ZYP1 (n = 7, 4, 10; Fig. S13-B to -D). Biochemical assays detected an interaction between ZmHOP2a and ZYP1, whereas no interaction with ASY1 was detected under the tested conditions. However, ZmHOP2a was largely excluded from chromosome regions fully loaded with ZYP1. Whether the ZmHOP2a–ZYP1 interaction occurs during meiosis and contributes to the stage-dependent localization of ZmHOP2a remains unresolved (Wang et al., 2023).

The mechanisms underlying the spatiotemporal regulation of this interaction remain elusive. Although γH2AX levels appeared normal, the repositioning of ZmHOP2a suggests that its function may be coupled to axis formation downstream of DSB formation. Future work should investigate whether post-translational modifications, such as those reported for yeast SC components (Storlazzi et al., 1996; Humphryes et al., 2013; Vrielynck et al., 2023), mediate the transient ZmHOP2a–ZYP1 association during maize meiosis.

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