ZmMTOPVIB is essential for double-strand break formation and bipolar spindle assembly during maize meiosis

The programmed formation of DNA double-strand breaks (DSBs) during early meiosis is catalyzed by SPO11, a conserved ortholog to the A subunit of an archaeal topoisomerase VI (TopoVI) which functions as a hetero-tetramer comprising two A and two B subunits. An essential role of the meiotic TopoVI B subunit (TopVIB) in DSB formation has been reported in mouse, Arabidopsis and rice. Very recently, rice MTopVIB was revealed to have an unexpected role in meiotic bipolar spindle assembly, highlighting multiple functions of MTopVIB during rice meiosis. In this work, the meiotic TopVIB in maize (ZmMTOPVIB) was characterized. The ZmmtopVIB mutant plants exhibited normal vegetative growth but male and female sterility. DSB formation is abolished in mutant meiocytes. Despite normal assembly of axial elements, synapsis was severely affected and homologous pairing was disrupted in mutants. Importantly, we showed that bipolar spindle assembly was also affected in ZmmtopVIB, resulting in triad and polyad formation. Overall, our results demonstrate that ZmMTOPVIB plays critical roles in DSB formation and homologous recombination. In addition, the newly-discovered function of MTOPVIB in bipolar spindle assembly is likely conserved across different monocots. One-sentence summary The dual roles of MTOPVIB in regulating meiotic DSB formation and bipolar spindle assembly are evolutionarily conserved in monocot plants.


96
In eukaryotes, faithful chromosome segregation during cell division is mediated by 97 spindle, i.e. a complex protein superstructure composed of microtubules and associated 98 proteins (Heald et al., 1996;Compton, 2000;Wittmann et al., 2001;Xue et al., 2019). 99 After nuclear envelope breakdown in plant meiosis, randomly-polarized microtubules 100 self-organize into bi-polar spindles during metaphase I (Vernos and Karsenti, 1995;Heald 101 et al., 1996;Gadde and Heald, 2004). A study in Zea mays (maize) has revealed that 102 microtubules form around the condensed bivalents, and then self-organized into a bipolar 103 spindles (Chan and Cande, 1998). In maize mutants defective in chromosome synapsis 104 and pairing, such as dsy1, dsy2, and afd1 meiocytes, spindle organization at metaphase I 105 form normally, indicating that bipolar spindle formation is independent of paired 106 kinetochores of bivalents (Chan and Cande, 1998). Therefore, it remains elusive that what 107 molecular pathways or regulatory factors are responsible for the process of bipolar 108 spindle assembly in maize. 109 Very recently, the rice MTopVIB analog (OsMTOPVIB) was found to play a key role  To identify a putative MTOPVIB gene in maize, the full-length amino acid sequence 127 of Arabidopsis MTOPVIB was used as a query to search in the maize genome database 128 (https://maizegdb.org/) by BLASTp analysis. We identified one candidate gene 129 (Zm00001d014728) with the highest similarity to Arabidopsis MTOPVIB (At1G60460). 130 By performing rapid amplification of cDNA ends (RACE), we obtained a 1,341 bp of 131 full-length ZmMTOPVIB cDNA sequence, which consists of 12 exons and 11 introns 132 ( Figure 1A). The amino acid sequences of MTOPVIB from 10 different plant species 133 obtained from NCBI were subject to phylogenetic analysis. The result revealed that two 134 distinct clades of MTOPVIB homologs represent genetic divergence of monocots and 135 dicots ( Figure S1). In addition, alignment of MTOPVIB protein sequences from 136 Arabidopsis, rice and maize revealed that their MTOPVIB proteins are largely conserved, 137 especially in three primary domains (GHKL, Small Domain and Transducer) ( Figure S2). 138 We then investigated spatio-temporal expression patterns of ZmMTOPVIB by quantitative 139 RT-PCR analyses and found that it was highly expressed in the developing tassel, 140 moderately expressed in embryo, ear and endosperm, and only weakly expressed in root, 141 stem and leaf ( Figure S3).

142
To characterize functions of MTOPVIB in maize, we obtained one mutant line 143 UFMu-07260 from the UniformMu population (Liu et al., 2016), which has a Mutator 144 7 inserted in the Zm00001d014728 gene. By PCR amplification and Sanger sequencing 145 using the Mutator and ZmMTOPVIB-specific primers (Table S1), we confirmed that the 146 Mutator transposon was inserted into the first intron of ZmMTOPVIB ( Figure 1B).

147
Although the insertion did not alter the ZmMTOPVIB expression ( Figure S3), it results in 148 aberrant splicing, leading to two major splice variants. The longer transcript appeared 149 much abundance relative to the shorter one, which is at the same size as in the wild type 150 ( Figure 1C). Sequence analyses revealed that the longer variant contains a fraction of  Figure 1D), which was predicted to abort splicing of the 156 8 th intron and to result in an in-frame premature stop codon (underlined taa) ( Figure 1D).

157
RT-PCR analysis showed a longer transcript in the mutant, confirming this intron 158 retention ( Figure 1E).

159
Both ZmmtopVIB mutants were completely male-sterile ( Figure 2A and Figure S4A), 160 and their anthers appeared withered at the flowering stage ( Figure 2B and Figure S4B).

161
Alexander Staining displayed that unlike the large, round, purple pollen grains of the wild 162 type ( Figure 2C and Figure S4C), mutant pollen grains were empty, shrunken, and unable 163 to stain ( Figure 2D and Figure S4D). In addition, mutant ears did not produce any seeds 164 when pollinated with pollen from wild type plants ( Figure 2E and Figure S4E). These 165 results indicate that defective ZmMTOPVIB causes both male and female sterility. Hence, 166 we named UFMu-07260 and EMS4-0742ae lines as ZmmtopVIB-1 and ZmmtopVIB-2 167 mutant alleles, respectively.     ZmMTOPVIB is required for meiotic bipolar spindle assembly in maize 286 The recently identified function of OsMTOPVIB in regulating meiotic spindle 287 assembly in rice prompted us to investigate if ZmMTOPVIB has the same role in maize 288 meiosis. To do this, we performed immunostaining using α-tubulin antibody in wild type 289 and ZmmtopVIB-1 meiocytes. α-Tubulin heterodimerizes and polymerizes with β-tubulin 290 to form microtubule walls (Blume et al., 2009;Gunning et al., 2015;Higgins et al., 2016). 291 We found that microtubule filaments in wild type meiocytes gradually extended and  with abnormal equatorial plates in telophase I ( Figure 7H and 7L, n=9).

306
To ascertain the outcomes of these abnormal spindle structures, we examined spore 307 products in wild type and ZmmtopVIB-1 mutant lines. In the wild type, we consistently  OsMTOPVIB are essential for DSB formation, it is conceivable that the processes of DSB 369 16 formation and bipolar spindle assembly may be coordinated. However, this speculation 370 was proven untrue upon discovery that bipolar spindles at metaphase I occur normally in 371 three rice mutants exhibiting defects in DSB formation, i.e., the pair2, Osspo11-1 and 372 crc1 mutants (Xue et al., 2019). Given these results, it is evident that meiotic spindle 373 assembly is independent of DSB formation. Additionally, bipolar spindle assembly also 374 seems to be uncoupled from homologous pairing since it was not perturbed in the dsy1, 375 dsy2, and afd1 mutants, three maize mutants defective in homologous pairing (Chan and 376 Cande, 1998). Therefore, the molecular mechanism that integrates these HR-related genes 377 in meiotic spindle assembly remains to be resolved.

378
As reported for the OsmtopVIB mutant, we observed a substantial proportion of 379 triads at telophase I and polyads at telophase II in ZmmtopVIB meiocytes, demonstrating 380 that maize ZmMTOPVIB is also required for meiotic bipolar spindle assembly. This      Chromosomes were marked with DAPI (blue), and microtubules were marked by 533 α-tubulin antibody (green). The red arrows pointed out the abnormal spindles and 534 equatorial plates. Scale bars = 10 μm.    (A-C) FISH in wild type meiocytes using a 5s rDNA (red) and telomere (green) probes at different stages. (D, E) Chromosome painting in wild type meiocytes using chromosome 3 (red) and chromosome 8 (green)specific probes. (F, G) FISH in ZmmtopVIB-1 meiocytes using a 5s rDNA (red) and telomere (green) probes at different stages.