A Hypomorphic Mutant of PHD Domain Protein Male Meiocytes Death 1

Meiosis drives reciprocal genetic exchanges and produces gametes with halved chromosome number, which is important for the genetic diversity, plant viability, and ploidy consistency of flowering plants. Alterations in chromosome dynamics and/or cytokinesis during meiosis may lead to meiotic restitution and the formation of unreduced microspores. In this study, we isolated an Arabidopsis mutant male meiotic restitution 1 (mmr1), which produces a small subpopulation of diploid or polyploid pollen grains. Cytological analysis revealed that mmr1 produces dyads, triads, and monads indicative of male meiotic restitution. Both homologous chromosomes and sister chromatids in mmr1 are separated normally, but chromosome condensation at metaphase I is slightly affected. The mmr1 mutant displayed incomplete meiotic cytokinesis. Supportively, immunostaining of the microtubular cytoskeleton showed that the spindle organization at anaphase II and mini-phragmoplast formation at telophase II are aberrant. The causative mutation in mmr1 was mapped to chromosome 1 at the chromatin regulator Male Meiocyte Death 1 (MMD1/DUET) locus. mmr1 contains a C-to-T transition at the third exon of MMD1/DUET at the genomic position 2168 bp from the start codon, which causes an amino acid change G618D that locates in the conserved PHD-finger domain of histone binding proteins. The F1 progenies of mmr1 crossing with knockout mmd1/duet mutant exhibited same meiotic defects and similar meiotic restitution rate as mmr1. Taken together, we here report a hypomorphic mmd1/duet allele that typically shows defects in microtubule organization and cytokinesis.


Introduction
Most higher plants, especially for angiosperms, have undergone at least one round of whole genome duplication (WGD) in evolution [1,2]. Formation and fusion of diploid or polyploid gametes are considered the primary route to plant polyploidization [3,4]. Alterations in one or more meiosis processes, including omission of meiotic cell cycle, defective chromosome segregation, spindle misorientation, and/or incomplete meiotic cytokinesis, are the common mechanisms leading to the generation of unreduced gametes through restitution of meiotic cell division [5][6][7][8][9][10]. In concern of the fundamental significance and practical utilization for polyploid crop breeding, it is of particular importance to uncover the genetic factors involved in unreduced gamete formation.
In Arabidopsis, at least 23 genes have been identified, which, in case of dysfunction, cause meiotic restitution (Table 1). Typically, functional mutations of the genes that control Genes 2021, 12, 516 2 of 14 meiotic cell cycle transition, such as OSD1/GIG1 and TAM/CYCA1;2, terminate the meiosis program prematurely, thereby inducing the production of 2n eggs and 2n spores [11]. Moreover, alterations of the three-dimensional positioning of spindles may result in unreduced gamete formation. The mechanism asserting the perpendicular position of the spindles is lost in parallel spindle1 (ps1) and jason mutants, which results in partial or complete fusion of the spindles [12,13]. Male meiotic cytokinesis in Arabidopsis is regulated by a mitogenactivated protein kinase (MAPK) signaling cascade composed of TES/STUD/AtNACK2-ANPs-MKK6/ANQ-MPK4. Loss of function of any member in this module leads to defects in male meiotic cytokinesis and the formation of pollen grains with an increased ploidy level [14][15][16][17][18]. Remarkably, the chromatin regulator Male Meiocyte Death 1 (MMD1/DUET), a PHD-finger protein that reads and binds with H3K4methylation sites, plays an important role in regulating multiple processes in Arabidopsis male meiosis [1,[19][20][21]. MMD1/DUET regulates the expression of meiotic genes, including TDM1, JASON, and CAP-D, 3, through binding to the H3K4me2/3 sites in the promoter regions [22,23]. The null mmd1/duet mutant displays chromosome de-condensation at metaphase I, altered meiosis progression, irregular spindle organization at anaphase II, and aberrant phragmoplast formation at telophase II, which lead to meiocyte cell death, meiotic restitution, and impaired plant fertility [22][23][24][25]. In search of genetic factors that contribute to ploidy consistency in meiosis, we previously performed a forward genetic screen of ethyl methanesulfonate (EMS)-mutagenized Arabidopsis thaliana Col-0 plants for mutants that produce over-sized pollen grains [13]. Here, the mutant male meiotic restitution 1 (mmr1) is described to consistently produce a relatively small fraction of unreduced pollen grains. The mmr1 mutant undergoes meiotic restitution and produces diploid and/or polyploid microspores. Meanwhile, mmr1 displays a mildly impacted chromosome condensation at metaphase I. Furthermore, the organization of spindles and phragmoplast in late meiosis II meiocytes of mmr1 are interfered, which results in defective cytokinesis. The causative point mutation in mmr1 was mapped to the third exon of MMD1/DUET within the conserved PHD domain. Overall, we here describe an allelic and hypomorphic mutant of Arabidopsis chromatin regulator MMD1/DUET.

Plant Materials and Growth Conditions
Arabidopsis thaliana wild-type accession Col-0 was used in this study. EMS-mutagenized Col-0 seeds were obtained from the Nottingham Arabidopsis Stock Centre. Arabidopsis pWOX2::CENH3-GFP [43] and mmd1/duet [22] were previously described. Seeds were germinated on K1 medium for 6 to 8 days, and seedlings were transferred to soil and cultivated in growth chambers at 12 h light/12 h night, 20 • C, and 70% humidity. Upon bolting, the photoperiod was changed to a 16 h day/8 h night regime.

Cytology and Microscopy
Pollen 4',6-diamidino-2-phenylindole (DAPI) staining, callosic cell wall staining, and analysis of the male meiotic products (tetrad-stage analysis by orcein and DAPI staining) were performed as described [21]. Sperm formation and chromosome counting was performed using the fluorescent marker pWOX2::CENH3-GFP and pMGH3::H2B-GFP lines. Spores were released in a 0.05 M phosphate buffer (pH 7.0) containing 0.5% Triton X-100 (v/v). Meiotic chromosomes were visualized following meiotic chromosome spreading and microtubules using immunostaining as described [21,44]. The assessment of unreduced pollen and microspores was performed by comparing the size to the haploid pollen (diameter >28 µm) or by counting the number of nuclei [45]. Bright-field and fluorescence microscopy were performed using an Olympus IX81 inverted fluorescence microscope equipped with an X-Cite Series 120Q UV lamp and an Olympus XM10 camera. Bifluorescent images and Z-stacks were processed using ImageJ. Brightness and contrast settings were adjusted using Photoshop CS6.

Identification of the Causative Mutation
The causative mutation in mmr1 was identified through bulk segregate analysis of progeny from a Col-0/Ler hybrid. The microsporogenesis of the F1 progenies obtained by homozygous mmr1 plants (female) crossing with Ler plants (male) were checked under microscope, and the genotype of the F1 progenies was examined by PCR using the primers listed in Supplementary Table S1. F2 population was collected by F1 selfing. Then, 1100 F2 individuals were checked and the samples showing meiotic restitution/samples phenocopied wild-type plants = 1:3, confirming that the meiotic restitution in mmr1 is caused by a single recessive mutation. F2 plants showing more than 5% oversized pollen grains were selected and genomic DNA was isolated and pooled for sequencing.

mmr1 Undergoes Meiotic Restitution
The formation of unreduced microspores suggests that meiotic restitution occurred in the microsporogenesis of mmr1. We hence analyzed and quantified tetrad-stage meiocytes in mmr1 using orcein staining. Wild-type plants consistently generated balanced tetrads that contained four haploid spores with each harboring one nucleus (Figure 2A,B). However, mmr1 plants were found to produce approximately 35.0% meiotic restituted products (Figure 2A) with 19.9% triads (Figure 2A,E), 8.7% unbalanced dyads (Figure 2A,D), 4.6% balanced dyads (Figure 2A,C) and 1.9% monads (Figure 2A,F), respectively. DAPI staining of tetrad-stage meiocytes confirmed the formation of dyad and triad with more than one nucleus per spore, which represented occurrence of meiotic restitution in the mmr1 mutant ( Figure 2G The formation of unreduced microspores suggests that meiotic restitution occurred in the microsporogenesis of mmr1. We hence analyzed and quantified tetrad-stage meiocytes in mmr1 using orcein staining. Wild-type plants consistently generated balanced tetrads that contained four haploid spores with each harboring one nucleus ( Figure  2A,B). However, mmr1 plants were found to produce approximately 35.0% meiotic restituted products (Figure 2A) with 19.9% triads (Figure 2A,E), 8.7% unbalanced dyads (Figure 2A,D), 4.6% balanced dyads (Figure 2A,C) and 1.9% monads (Figure 2A,F), respectively. DAPI staining of tetrad-stage meiocytes confirmed the formation of dyad and triad with more than one nucleus per spore, which represented occurrence of meiotic restitution in the mmr1 mutant ( Figure 2G   Meiotic spreading was performed to monitor the chromosome behaviors in mmr1 meiosis ( Figure 3). In wild-type plants, homologous chromosomes were fully paired at pachytene ( Figure 3A), and five pairs of bivalents occurred at diakinesis ( Figure 3B). No obvious defect was observed in mmr1 meiocytes at these stages, indicating normal homolog synapsis and crossover formation ( Figure 3I,J). At metaphase I, most bivalents in the wild-type showed chromosomes under tension (ratio of bivalents with tension/bivalents without tension = 2.5), aligned at the equatorial plate ( Figure 3C). In mmr1, however, more bivalents displayed an aberrant shape at metaphase I and lacked the typical thin threads at either side that result from the pulling force at the kinetochore (bivalents with tension/bivalents without tension = 1.5) ( Figure 3K). The minor difference of metaphase I chromosomes suggested that the mmr1 mutation has a mild impact on chromosome condensation. mmr1 chromosomes behaved normally from the interkinesis to metaphase II stages as control ( Figure 3D These data indicate that although both homologous chromosomes and sister chromatids in the mmr1 mutant were able to segregate, positioning of haploid chromosome sets at telophase II was somehow interfered, which implied an alteration in proper spindle orientation and/or cytokinesis.

Meiotic Cytokinesis Is Defective in mmr1
To determine whether meiotic restitution in mmr1 was caused by a defect in cytokinesis, we analyzed meiotic cell walls using aniline blue staining that specifically marks callose. Tetrads in the wild-type plants generated a cross-shaped cell wall configuration, indicating four haploid spores ( Figure 4A). In contrast, mmr1 meiocytes were observed to have one or more cell walls omitted, displaying balanced dyads ( Figure 4B), unbalanced dyads ( Figure 4C,E) or triads ( Figure 4D,F). Moreover, interrupted cell walls were occasionally observed ( Figure 4E,F, see red arrows). These figures indicate that mmr1 is defective for generating complete meiotic cell walls during cytokinesis.

Meiotic Cytokinesis Is Defective in mmr1
To determine whether meiotic restitution in mmr1 was caused by a defect in cytokinesis, we analyzed meiotic cell walls using aniline blue staining that specifically marks callose. Tetrads in the wild-type plants generated a cross-shaped cell wall configuration, indicating four haploid spores ( Figure 4A). In contrast, mmr1 meiocytes were observed to have one or more cell walls omitted, displaying balanced dyads ( Figure 4B), unbalanced dyads ( Figure 4C,E) or triads ( Figure 4D,F). Moreover, interrupted cell walls were occa- sionally observed ( Figure 4E,F, see red arrows). These figures indicate that mmr1 is defective for generating complete meiotic cell walls during cytokinesis.

Altered Spindle Orientation and Phragmoplast Formation in mmr1
Construction of meiotic cell walls relies on the organization of the microtubular cytoskeleton surrounding chromosomes or nuclei [21]. Immunostaining of α-tubulin was therefore applied to elucidate whether the meiotic cytokinesis defects in mmr1 was caused by any alteration in microtubules. From prophase I to metaphase II, mmr1 did not show any obvious difference in microtubular network formation, as in wild-type plants ( Figure 5B,D,F,H,J, Col-0; C,E,G,I,K, mmr1). Successfully generated spindles at metaphase I and II supported the observation that both chromosome segregation cycles were not influenced in mmr1 ( Figure 5E,K). However, at telophase II, the orientation of phragmoplast in mmr1 displayed either parallel or tripolar configuration ( Figure 5L, Col-0; M,N, mmr1; Supplementary Figure S5A,B). In wild-type and most mmr1 tetrad stage meiocytes, radial microtubule arrays (RMAs) were organized into mini-phragmoplasts between the separated nuclei, contributing to tetrad formation ( Figure 5O, Col-0; P, mmr1). Approximately 48.0% tetrad stage meiocytes in mmr1, however, showed omission of one or more RMAs between the separated nuclei ( Figure 5A Figure S5G, mmr1). Meanwhile, the microtubules of RMAs were also less compacted than in Col-0 ( Figure 5O, Col-0; P-S and Supplementary Figure S5C-H, mmr1). Taken together, these findings re-

Altered Spindle Orientation and Phragmoplast Formation in mmr1
Construction of meiotic cell walls relies on the organization of the microtubular cytoskeleton surrounding chromosomes or nuclei [21]. Immunostaining of α-tubulin was therefore applied to elucidate whether the meiotic cytokinesis defects in mmr1 was caused by any alteration in microtubules. From prophase I to metaphase II, mmr1 did not show any obvious difference in microtubular network formation, as in wild-type plants ( Figure 5B,D,F,H,J, Col-0; C,E,G,I,K, mmr1). Successfully generated spindles at metaphase I and II supported the observation that both chromosome segregation cycles were not influenced in mmr1 ( Figure 5E,K). However, at telophase II, the orientation of phragmoplast in mmr1 displayed either parallel or tripolar configuration ( Figure 5L Figure S5A,B). In wild-type and most mmr1 tetrad stage meiocytes, radial microtubule arrays (RMAs) were organized into mini-phragmoplasts between the separated nuclei, contributing to tetrad formation ( Figure 5O, Col-0; P, mmr1). Approximately 48.0% tetrad stage meiocytes in mmr1, however, showed omission of one or more RMAs between the separated nuclei ( Figure 5A; Supplementary Table S2), which generated triads (36.3%) ( Figure 5S; Supplementary Figure S5G Figure S5G, mmr1). Meanwhile, the microtubules of RMAs were also less compacted than in Col-0 ( Figure 5O, Col-0; P-S and Supplementary Figure S5C-H, mmr1). Taken together, these findings revealed defective spindle orientation, and formation and/or organization of phragmoplasts at meiosis II in the mmr1 mutant.
12, x FOR PEER REVIEW 10 of 15 vealed defective spindle orientation, and formation and/or organization of phragmoplasts at meiosis II in the mmr1 mutant.

mmr1 Carries a Point Mutation in the PHD-Finger Domain of MMD1/DUET
To identify the causative mutation in the mmr1 mutant, we performed bulk segregant analysis combined with whole genome sequencing. F1 progenies from the intercrossing between mmr1 mutant and wild-type Landsberg erecta (Ler) was genotyped using simple sequence length polymorphism (SSLP) primers to check Col0/Ler heterozygosity (Supplementary Figure S6). All of the F1 individuals displayed normal male meiotic cytokinesis and produced normal-sized male gametes (Supplementary Figure  S7A-C), suggesting a recessive mutation in mmr1. The phenotypic screening of 1100 F2 Col/Ler descendants resulted in 250 individuals producing unreduced microspores, and DNA from individuals were pooled and sequenced. A 26 Mb region on chromosome 1 was enriched for Col-0 SNPs (Supplementary Figure S8). Several genes within this region carried mutations, but the candidate gene MMD1/DUET (AT1G66170) was the only meiosis-specific one. mmr1 was found to carry a single C-to-T transition at the genomic position 2168 bp from the start codon of MMD1/DUET, leading to an amino acid change G618D ( Figure 6A; Supplementary Table S3). The G618D amino acid change was located within the plant homeodomain (PHD) of MMD1/DUET, which has been found in many chromatin regulatory factors and conserved in histone binding proteins ( Figure 6B) [24]. A complementation test was performed by crossing homozygous mmr1 mutant with homozygous mmd1 null mutant using mmr1 as the pollen donor. All checked F1 progenies produced unreduced microspores and pollen, and showed defects in meiotic cytokinesis ( Figure 6C-G, Col-0; H-O, mmr1/mmd1 F1 progenies), which indicates that mmr1 is allelic to MMD1/DUET. Since mmr1 produced a small population of unreduced microspores, it is thus a hypomorphic mmd1/duet allele.

mmr1 Carries a Point Mutation in the PHD-Finger Domain of MMD1/DUET
To identify the causative mutation in the mmr1 mutant, we performed bulk segregant analysis combined with whole genome sequencing. F1 progenies from the intercrossing between mmr1 mutant and wild-type Landsberg erecta (Ler) was genotyped using simple sequence length polymorphism (SSLP) primers to check Col0/Ler heterozygosity (Supplementary Figure S6). All of the F1 individuals displayed normal male meiotic cytokinesis and produced normal-sized male gametes (Supplementary Figure S7A-C), suggesting a recessive mutation in mmr1. The phenotypic screening of 1100 F2 Col/Ler descendants resulted in 250 individuals producing unreduced microspores, and DNA from individuals were pooled and sequenced. A 26 Mb region on chromosome 1 was enriched for Col-0 SNPs (Supplementary Figure S8). Several genes within this region carried mutations, but the candidate gene MMD1/DUET (AT1G66170) was the only meiosis-specific one. mmr1 was found to carry a single C-to-T transition at the genomic position 2168 bp from the start codon of MMD1/DUET, leading to an amino acid change G618D ( Figure 6A; Supplementary Table S3). The G618D amino acid change was located within the plant homeodomain (PHD) of MMD1/DUET, which has been found in many chromatin regulatory factors and conserved in histone binding proteins ( Figure 6B) [24]. A complementation test was performed by crossing homozygous mmr1 mutant with homozygous mmd1 null mutant using mmr1 as the pollen donor. All checked F1 progenies produced unreduced microspores and pollen, and showed defects in meiotic cytokinesis ( Figure 6C-G, Col-0; H-O, mmr1/mmd1 F1 progenies), which indicates that mmr1 is allelic to MMD1/DUET. Since mmr1 produced a small population of unreduced microspores, it is thus a hypomorphic mmd1/duet allele.

Discussion
In this study, we reported the identification of a recessive mutant mmr1 that consistently produces a small fraction (around 5%-10%) of unreduced pollen grains. The phenotype is caused by a single amino acid change in the PHD domain of MMD1/DUET, a chromatin regulator that is specifically expressed in male meiocytes and functions in male meiosis [24,25]. Ds transposon insertions in the exon of MMD1/DUET lead to MMD1/DUET null mutants, which display strong male sterility due to various defects in meiosis that culminate into meiocyte cell death [24,25]. Male meiosis in MMD1/DUET is initiated normally, forming meiocytes that progress up to pachytene as in the wild-type. During the subsequent steps in the meiosis program, two cycles of chromosome condensation take place. In mmd1/duet, diakinesis chromosomes are paired into bivalents and appear less compact and show inter-bivalent interactions [24,25,47], which were, however, not observed in mmr1. In mmr1, an alteration in chromosome structure was observed at metaphase I, when bivalents are under tension from kinetochore microtubule pulling forces. In the wild-type, the kinetochores are slightly pulled apart, which is evident from the thin DAPIstained threads perpendicular to the equatorial plane. These extensions are also absent, or at least far less pronounced, in null mmd1/duet [23]. In mmr1, the altered chromosome condensation in metaphase I is not as severe as in the null mmd1/duet mutant, suggesting that mmr1 is hypomorphic. The reduction in fertility of mmr1 is very mild compared to mmd1/duet, in line with the occurrence of cytological defects in only a subpopulation of the meiocytes and microspores. The mmr1 mutation therefore has a minor impact on the functioning of MMD1/DUET.
The G618D amino acid change in mmr1 is located within the PHD domain of MMD1/DUET, which forms an aromatic cage, required for the specific binding to H3K4me2 [22,23,47]. MMD1/DUET may bind a wider range of histone peptides, as a recombinant peptide encompassing the MMD1/DUET PHD finger (601 to 659) was shown to bind the histone peptides H3K4me2/me3, H3K9me2, and H3S10 [23]. The G618D amino acid change in the mmr1 allele introduces a negative charge in the PHD finger, modifying its electrostatic balance and likely changing the strength and/or specificity of histone binding. As mmr1 displays a hypomorphic phenotype, we speculate that the mmr1 allele has the same histone specificity but with slightly weaker binding affinity. The PHD domain is of critical importance to MMD1/DUET function, as PHD deletion mutants and selected PHD amino acid changes show the same sterility phenotype as a knockout line [23,47]. Andreuzzi et al. (2015) [22] reported on a hypomorphic MMD1/DUET PHD finger mutation that causes the formation of a low frequency of enlarged pollen grains (approximately 5%) reminiscent to mmr1 [22]. The reported hypomorphic mutant appeared to be defective specifically in meiosis II spindle and RMA organization [22]. Chromosome condensation, M II phragmoplast, and RMA organization are affected in mmr1, suggesting that the G618D amino acid change interferes with MMD1/DUET functioning during both meiosis I and II. The mild alteration in chromosome condensation at metaphase I in mmr1 suggests that the point mutation G618D is more related to MMD1/DUET function in phragmoplast organization.

Conclusions
MMD1/DUET has been shown to profoundly modulate gene expression in anthers and seems to do this differentially during early stage and late stage of meiosis [23]. Expression of the meiotic cell cycle regulator TDM and the meiosis spindle II organizer JASON is severely reduced in duet, in line with the defects in spindle position and cytokinesis [22]. MMD1/DUET also directly regulates the expression of the condensin genes CAP-D2, CAP-D3, CAP-H, and CAP-H2. The cap-d3 mutants show a male meiotic chromosome condensation phenotype such as mmd1/duet, indicating that the prophase I chromosome condensation phenotype is due the misexpression of CAP-D3 in MMD1/DUET [23]. Since mmr1 shows a very mild phenotype in metaphase I chromosome condensation and a low frequency of meiotic restitution, we speculate that a reduction in the expression of CAP-D3 and Jason is minor and only occasionally drops below a threshold to generate the observed defects in chromosome condensation and cytokinesis. The isolated mmr1 mutant in this report thus provides a tool to analyze the function of regions within the PHD domain in MMD1/DUET with a specific correlation to telophase II and cytokinesis.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.