Introduction

The correct segregation of sister chromatids is essential for mitotic cell stabilization and to generate haploid gametes during meiosis. The cohesin complex is a conserved multi-subunit protein system that guarantees the proper segregation of sister chromatids in mitosis and across living organisms. This complex is formed by four core subunits, including the structural maintenance of chromosomes (SMC) subunits SMC1 and SMC3, and the sister chromatid cohesin (SCC) subunits SCC1 and SCC3 (Bolanos-Villegas et al., 2017; Moronta-Gines et al., 2019). The cohesin complex forms a ring encircling the sister chromatids and ensures genomic stability during DNA replication (Higashi et al., 2020). In addition, cohesin also participates in the repair of DNA double-strand breaks (DSB), homologous pairing, construction of the synaptonemal complex (SC), orientation of kinetochores, and regulation of gene expression (Nasmyth and Haering, 2009).

Cohesin is established during the S phase, and includes arm and centromeric cohesin. Arm cohesin ensures sister chromatids bond together during mitotic prophase. Centromeric cohesin persists until the metaphase-to-anaphase transition, and is vital to resist the force exerted by spindle microtubules, allowing for the accurate segregation of sister chromatids to opposite poles of the cell. The dissociation of cohesin generally occurs in two steps, including through the dissociation of cohesin on chromosome arms during prometaphase, followed by the degradation of cohesin at the centromeres at the onset of anaphase (Ishiguro and Watanabe, 2007; Watanabe, 2005). This allows sister chromatids to segregate in mitosis.

Meiosis contains a single round of DNA replication, but, in contrast to mitosis, is followed by two successive chromosome divisions. Nonetheless, while the loading mechanism of cohesin is very similar to the analogous process during mitosis, the cohesin degradation during meiosis occurs in two steps: from chromosome arms during the first meiotic division, and from the centromeres during the second meiotic division (Mercier et al., 2015). During meiosis I, homologous chromosomes are linked by chiasmata that separate at metaphase I. At this stage, cohesin dissociates from chromosome arms while the two sister chromatids remain closely together because centromeres cohesin are preserved. Subsequently, the sister chromatids separate at anaphase II due to full cohesin disassociation at the chromosome arms and centromeres. During this process, sister chromatids cohesin is protected by shugoshin from cleavage by separase (Wang et al., 2012; Wang et al., 2011), which determines the correct separation of sister chromatids during meiosis.

Notably, the cohesin complex plays additional functional roles in meiosis. Specifically, cohesin on chromosome arms is crucial for normal progression of meiosis I, while centromeric cohesin plays a more prominent role in meiosis II by geometrically orientating the sister chromatids. During meiosis I, homologous chromosomes are segregated in opposite directions to halve the chromosome number, but sister chromatids remain temporarily mono-oriented and move to the same pole. In meiosis II, sister chromatid pairs become bi-oriented and separate towards opposite spindle poles through similar mechanisms as those observed in mitosis (Schvarzstein et al., 2010). In this process, centromeric cohesin plays a crucial role in establishing kinetochore geometry and thus determining sister kinetochore orientation (Gryaznova et al., 2021). In addition, the cohesin complex impacts the formation of chromosome axial elements (AE) and the assembly of the synaptonemal complex (SC) (Agostinho et al., 2016; Fujiwara et al., 2020; Llano et al., 2012). Besides, the cohesin complex regulates gene expression in eukaryotes, acting as a transcriptional co-activator in humans that is recruited by sequence-specific transcription factors (Casa et al., 2020; Kagey et al., 2010; Lara-Pezzi et al., 2004; Remeseiro et al., 2012). Finally, the cohesin complex is now emerging as a key player in DSBs repair pathway choice and efficiency. In yeast, cohesin and DMC1 may interactively modulate homologous recombination partner choice, with program-appropriate effects (Hong et al., 2013; Phipps and Dubrana, 2022; Sanchez-Moran et al., 2007).

To date, there were many researches on cohesin subunits, which had different functions throughout the cell cycle. In yeast, SCC1 ensures sister chromatid cohesin in mitosis and is associated with chromosomes during the S phase, dissociating during the metaphase-to-anaphase transition. The expression of SCC1 gradually decreases at the beginning of meiosis, whereby SCC1 likely plays a minor role during meiosis (Michaelis et al., 1997). Instead, REC8 is a special meiotic-specific cohesin component that replaces SCC1 in yeast (Klein et al., 1999; Watanabe and Nurse, 1999) and which is expressed in leptotene and decreases after pachytene (Shao et al., 2011). REC8 also plays a crucial role establishing sister kinetochore orientation in yeast, mice and plants (Chelysheva et al., 2005; Ogushi et al., 2021; Toth et al., 2000). In addition, rec8 mutants in flowering plants exhibit sticky chromosomes, which severely affects the localization of other key meiotic proteins (Cai et al., 2003; Chelysheva et al., 2005; Shao et al., 2011). Moreover, SCC3-SCC1 subcomplex is necessary for cohesin loading and the establishment of sister chromatid cohesin in mitosis in yeast (Li et al., 2018a; Orgil et al., 2015). SCC3 also being responsible for maintaining centromeric orientation during meiosis in Arabidopsis (Cai et al., 2003; Chelysheva et al., 2005; Kulemzina et al., 2012). Taken together, these results indicated that cohesin is crucial for the stability of chromosome structure in mitosis and meiosis. However, does cohesin have other functions? such as participating in homologous recombination and DSBs repair process? Also, the mechanisms through which SCC3 regulates the assembly of sister chromatid cohesin in mitosis and its function in meiosis were not well investigated. SCC3 and REC8 are both meiotic cohesin, and what is the difference between them?

Here, we further investigated SCC3 functions, demonstrating its involvement in mitosis and meiosis. We observed the replication process of chromosome 12 during interphase confirming that SCC3 stabilizes chromosome replication process both in mitosis and meiosis, which is crucial for sister chromatids cohesin. Furthermore, we investigated the process of cohesin loading and degradation on chromosomes by using the antibody of SCC3 on chromosomes during mitosis and meiosis. We also show the localization of SCC3 is regulated by REC8. Meanwhile, other meiotic proteins (COM1, DMC1, RAD51, PAIR2 and PAIR3) failed to localize in rec8. That is, REC8 is a fundamental meiotic protein which is prerequisite for the localization of other meiotic proteins. However, the meiotic components (DMC1, RAD51, ZIP4, HEI10 and ZEP1) could be present in scc3 but apparently making no functional contribution to recombination and synapsis indicating that SCC3 participates in homologous pairing, synapsis, recombination progress and CO formation. More importantly, we found meiotic DSBs were well repaired in scc3, probably using sister chromatids as a template, which means SCC3, as a member of cohesin complex, could inhibit inter-sister chromatids repair during meiosis. Finally, we explore different roles of SCC3 and REC8 in meiosis. All findings are integrated into a coherent model.

Results

SCC3 causes both vegetative and reproductive growth defects

We performed a BLAST search in the Rice Genome Annotation Project database (http://rice.plantbiology.msu.edu/cgibin/gbrowse/rice) and found a single hypothetical homologue of SCC3 proteins (A. thaliana SCC3, M. musculus STAG3 and H. sapiens SA1). This candidate protein is encoded by the Os05g0188500 gene and shares the highest similarity with AtSCC3. The full-length cDNA of SCC3 is 3755 nucleotides long and comprises 22 exons and 21 introns. SCC3 contains a 3345-nucleotide-long ORF that encodes a 1116aa protein. In higher eukaryotes, SCC3 is an evolutionary conserved protein that contains a highly conserved STAG domain (140-239aa), as predicted by SMART (Figure S1A). Multiple alignments of full-length SCC3 protein sequences and its homologs in other plants revealed the STAG domain is highly conserved across both monocotyledons and dicotyledons (Figure S1B). Homology modeling indicated SCC3 protein structures are strongly conserved in different species, which also possess similar STAG domains (Figure S2A). Additionally, a phylogenetic tree was constructed with full-length SCC3 amino acid sequences that confirmed this protein is conserved in eukaryotes (Figure S2B). We also did multiple sequence alignments of these proteins used in the evolutionary tree analysis (Figure S3).

A previous study in Arabidopsis (Chelysheva et al., 2005) identified a T-DNA insertion at the boundary between intron 5 and exon 6 of SCC3 that caused lethality, whilst a weak allele was able to survive albeit with some developmental defects. To clarify SCC3 function in rice, we used the CRISPR-Cas9 system to generate three transgenic lines (Figure S4). The first allele (scc3-1) contains a frameshift “GA” deletion in exon 2 and the second allele (scc3-2) contains a frameshift “ACCGA” deletion in exon 11. We tested for allelism between these two scc3 mutants by crossing scc3-1^+/- (male) and scc3-2^+/- (female). Of the 78 F1 plants, 18 were scc3-1^+/-, 26 were scc3-2^+/-, 34 were wildtype for both loci. Thus, these two mutants are allelic and heterozygous scc3-1/scc3-2 is lethal. We were unable to isolate homozygous mutants in these two allelic progenies (n=128, 87 plants were scc3-1^+/- and 41 were wildtype; n=116, 80 plants were scc3-2^+/- and 36 were wildtype). These results suggested that scc3-1 and scc3-2 mutants were both embryo-lethal.

In addition, we obtained a weak transgenic line (scc3) containing a frameshift “T” insertion in exon 19 leading to premature termination of translation (Figure S4). We found the scc3 weak mutant exhibits abnormal vegetative growth (Figures 1A and 1B), and thus proceeded with further investigation of its other phenotypic effects, which revealed scc3 decreases plant height, tiller number and panicle length (Figures 1C and S5B-S5D). To investigate the patterns of SCC3 expression, we employed RT-PCR in various tissues and found the gene is ubiquitously expressed, particularly in roots, leaves and small panicles (Figure S5A). Accordingly, the weak mutant of SCC3 causes severe vegetative growth defects in rice plants.

Figure 1.

To elucidate whether SCC3 is involved in meiosis, we performed cytological observation of anthers stained with 1% I₂-KI, and found almost all pollen grains were shrunken and inviable (Figures 1D and S5E). Our results also showed scc3 flowers did not set seeds when pollinated with wild-type pollen, suggesting the mutant was sterile. We further observed heterozygous scc3 plants produced progenies with a segregation ratio of 3:1 (normal: dwarf and sterile), indicating the scc3 mutation is recessive and monogenic. These data suggest that scc3 weak mutation dramatically interferes with plant vegetative and reproductive growth, which may be relevant to mitosis and meiosis.

SCC3 is required for sister chromatid cohesin during mitosis

To determine whether SCC3 is involved in sister chromatid cohesin in mitosis, we observed chromosome behavior in root tip cells of scc3 and wildtype. DNA replicates and folds into an ordered structure during interphase in wildtype plants (Figure 1E). After this, the chromosomes condensed and formed short thread-like chromatids during prophase, which developed into further condensed sister chromatids in prometaphase that were aligned on the equatorial plate before separating to different poles of the cell.

In contrast, scc3 exhibited “X”-shaped chromosomes with distant arms and telomeres in prometaphase (Figures 1E and 1G), indicating a loss of cohesin between sister chromatids. Approximately two-thirds of scc3 root tip cells (70.6%, n=88) contained partially or completely separated sister chromatids (Figure 1H), which were loosely arranged on either side of the equatorial plate during the anaphase-to-telophase transition (Figure 1E), demonstrating the sister chromatids separated in advance as they were pulled to the spindle poles of the cell. But we did not observe any aneuploidy in anaphase, because mitotic tubulin was attached to centromeres of sister chromatids during prometaphase, and sister chromatids were still together at this time in scc3, which guarantee sister chromatids separated equally.

Furthermore, we monitored the distance between centromeres using FISH probes with centromere-specific tandem repetitive sequence CentO in both wildtype and scc3 somatic cells during prometaphase. We found the distance between sister centromeres also dramatically increased in scc3 (Figure 1F, n=25). However, we also noted that, despite the distance between sister chromatids increased in scc3 weak mutants, sisters were still observed side-by-side during prometaphase, suggesting that there may be other cohesin proteins to maintain sister chromatids associated. Our results demonstrated SCC3 is required for cohesin between sister telomeres, centromeres and arms during mitosis.

To explore whether SCC3 affects the replication process of sister chromatids, we performed full-length FISH assays to monitor the dynamic phenotype of chromosome 12 during interphase. In the wildtype, sister chromatids were replicating that could be observed during interphase (Figure 2A). Subsequently, two chromosome clumps formed during prophase and developed into high-order linear chromosomes during prometaphase. During this time, two sister chromatids seemed overlapped and condensed into a short stick. However, in scc3 an obvious chromosomes fragmentation was observed during interphase, which means the replicating chromatids are abnormally organized. During prophase, chromosome 12 were still into a lump and unable to condense into a stick phenotype. In prometaphase, these abnormal chromosomes finally form loosely-connected sister chromatids (Figure 2A). These observations indicated that SCC3 is involved in the replication process of sister chromatids, which may be implicated as a regulator of the genome, influencing chromatin composition and dynamics, and crucially genome organization through folding chromosomes.

Figure 2.

To investigate the localization of SCC3 during mitosis, we generated a mouse polyclonal antibody against C-terminal and performed immunostaining of root tip wildtype cells (Figure 2B). We noted no SCC3 signal was present in scc3 root tip cells, confirming the specificity of the SCC3 antibody utilized in mitosis (Figure S6A). We found SCC3 was uniformly distributed in the cells during interphase in most cases. From interphase to prophase, as chromosome replication completed, the centromere signals became brighter and SCC3 signals also become denser and brighter. SCC3 eventually converged into intact chromosomes at prometaphase. From metaphase to telophase, SCC3 signal disappeared (Figure 2B). These observations suggested that SCC3 may enhance the interaction with DNA during the transition from interphase to prophase, and dissociated from chromosomes after prometaphase.

SCC3 acts as an axial element during meiosis

REC8 is a special axial protein indicating meiotic progression (Shao et al., 2011). To evaluate SCC3 localization in meiosis, we performed dual-immunolocalization between SCC3 and REC8 in wildtype meiocytes. REC8 signals were initially detected as foci in the nucleus at leptotene, while SCC3 was detectable simultaneously (Figure 3A). From leptotene to zygotene, SCC3 signals were detected as similar elongated foci that formed filamentous structures on the chromosomes. During pachytene and diplotene, constant SCC3 signals were distributed along chromosome axes (Figure 3A). At diakinesis, axial SCC3 signals became diffuse and centromeric signals gradually became clear. At metaphase I, only centromeres SCC3 antibody signals remained (Figure 3B). However, REC8 signals were difficult to detect from diakinesis to metaphase I (Figure S7). In order to further clarify the localization pattern between SCC3 and REC8, we performed super-resolution imaging (SIM) on wildtype meiocytes, which showed REC8 and SCC3 were parallel and colocalized forming parallel axial elements (Figure 3C). In addition, central elements ZEP1 formed two parallel lines surrounded by SCC3 signals (Figure 3D). We also noted no SCC3 signal was present in scc3 meiocytes, confirming the specificity of the SCC3 antibody utilized in meiosis (Figure S6B).

Figure 3.

SCC3 is required for sister chromatid cohesin in early meiosis

The interphase of meiosis is similar to mitosis, and sister chromatids replicate during interphase and are linked together by cohesin. To determine whether SCC3 is involved in sister chromatid cohesin during meiosis, we observed chromosome behaviors of meiocytes in scc3 and wildtype before leptotene. Cytological phenotypes of meiosis initiation in rice have been reported in detail (Zhao et al., 2018). The first meiotic stage that can be distinguished is preleptotene, and sister chromatids should have completed replication during this time. In preleptotene, the meiocytes chromosomes were discrete and partially contracted during preleptotene (Figure 4A), and subsequently formed thin threads in the wildtype at leptotene. In scc3, chromosomes at preleptotene exhibited dispersed appearance with blurred outlines. During leptotene, chromosomes maintained a loose shape and no filamentous threads were present. These observations indicate SCC3 participates in sister chromatids replication process in early meiosis.

Figure 4.

We then examined chromosome behavior in rec8 single mutants and scc3 rec8 double mutants at preleptotene (Figure 4A). Unexpectedly, rec8 exhibited elongated chromosomes similar to the wildtype, whereas scc3 rec8 exhibited diffused chromosomes similar to scc3. These results indicate REC8 does not affect the early replication process of sister chromatids, and the chromosome defects in scc3 rec8 double mutant may be caused by the mutation of SCC3. In addition, we performed full-length FISH assays to monitor chromosome 12 at preleptotene (Figure 4B), and found that replicated chromosome 12 can form short rod-shaped threads in both wildtype and rec8, but appears as a diffuse structure in scc3 and scc3 rec8 mutants due to abnormal replication process. These observations are consistent with DAPI staining results, further demonstrating SCC3 affects sister chromatid replication process in early meiosis.

Homologous chromosome pairing and synapsis are disturbed in scc3

To study the causes of sterility in scc3, we investigated male meiotic chromosome behavior of wildtype and scc3 meiocytes. In the former, homologous chromosomes paired at early zygotene and fully synapsed at pachytene. Moreover, during metaphase I, 12 bivalents were orderly arranged on the equatorial plate, with homologous chromosomes separating at anaphase I (Figure 5A). In contrast, scc3 chromosome behavior showed no differences at zygotene, with meiotic defects becoming apparent at pachytene, when most chromosomes dispersed as single threads with incomplete homologous paring (Figure 5A). We identified 24 univalents during diakinesis and metaphase I (n=161, 150 meiocytes have 24 univalents, 11 meiocytes have 1-2 bivalents), which were always pulled to opposite poles leading to sister chromatids separation at anaphase I. However, in spo11 mutants containing 24 univalents, sister chromatids were separated randomly or still linked together (Figure S8A). These observations show SCC3 determine the separation of sister chromatids during anaphase I, and is involved in homologous pairing, which could influence the normal process of synapsis.

Figure 5.

The telomere bouquet is a prerequisite for homologous pairing at early zygotene (Zhang et al., 2020). We performed a FISH assay to detect bouquet formation in wildtype and scc3 meiocytes using a telomere-specific probe (pAtT4). As expected, the wildtype telomeres were attached to a confined region, forming a typical bouquet on the nuclear envelope at zygotene. Interestingly, the formation of telomere bouquets also occurred normally in scc3 (Figure S8B). We then used 5S rDNA to monitor homologous pairing, and detected two overlapping 5S rDNA foci at pachytene in the wildtype, but two separated 5S rDNA foci in unpaired homologous chromosomes in scc3 (n=52; Figure S8C), confirming SCC3’s role in homologous pairing.

Homologous chromosomes align and then become closely juxtaposed by a proteinaceous structure known as the synaptonemal complex (SC), which is comprised of two parallel arrays of axial elements (AE), termed lateral elements (LE) prior to synapsis, that form the structural basis for the assembly of the SC (Ren et al., 2021). Since SCC3 is a LE component, we monitored the assembly of the SC through immunostaining using an antibody against ZEP1 in scc3 and wildtype meiocytes. In the wildtype, we observed ZEP1 gradually elongated and formed short linear signals during zygotene, which eventually extended along the paired homologous chromosomes at pachytene (Figure 5B). However, scc3 mutants displayed ZEP1 as a few punctate foci during zygotene. These abnormal structures were also present at pachytene (Figure 5B), suggesting SCC3 plays a crucial role in the assembly of the SC. In addition, we also examined the localization of other LEs (PAIR2 and PAIR3), and these proteins behave normally in scc3 (Figure S9).

SCC3 is essential for recombination progress and CO formation

Since homologous pairing was impaired in scc3 meiocytes, we wondered whether recombination was normal in scc3. γH2AX is a marker that is used as a proxy to monitor DNA DSBs. Therefore, we investigated the number of γH2AX foci to explore the DSB formation. There was no significant difference in the number of γH2AX foci between the wildtype and scc3 (265.4 ± 8.6, n=31 for scc3 meiocytes; 262.2 ± 7.2, n=20 for wildtype meiocytes) (Figure 6A). Additionally, we also examined the DSB resection factor (COM1), meiotic strand-invasion and exchange factors (RAD51, DMC1) and the Class I, interference-sensitive crossover (CO) marker (ZIP4) in both wildtype and scc3. Immunostaining results revealed that the number of COM1 foci (290.3 ± 8.8, n=26 for scc3 meiocytes; 293.8 ± 9.4, n=23 for wildtype meiocytes) were not significantly different between the wildtype and scc3 (Figures 6A and 6B). However, DMC1 (65.4 ± 17.1, n=25 for scc3 meiocytes; 281.2 ± 8.3, n=20 for wildtype meiocytes), RAD51 (55 ± 21.7, n=26 for scc3 meiocytes; 306 ± 11.9; n=24 for wildtype meiocytes) and ZIP4 (58 ± 17.9, n=23 for scc3 meiocytes; 316.6 ± 11.1, n=21 for wildtype meiocytes) foci were significantly decreased in scc3, suggesting that recombination progress and CO formation was disturbed in scc3. Previous studies revealed that HEI10 is a marker of Class I COs. In wide type, prominent HEI10 foci were observed (24.2 ± 3.8, n=25) at late pachytene (Figures 6A and 6B). By comparison, in scc3 most HEI10 foci were sparse and did not present as large bright signals. Taken together, these results suggested that CO maturation was disturbed in scc3. Although recombinant proteins could be localized in a small portion, the homologous recombination process was still greatly restricted.

Figure 6.

SCC3 inhibits inter-sister chromatids repair during meiosis

It is worth noting that DSBs formation was normal in scc3, and inter-homolog strand-invasion is destabilized which leads to the defects in CO formation. Nevertheless, in scc3, the meiotic DSBs are reliably repaired as there is no chromosome fragmentation, most likely using sister chromatid as a repair template, leading to 24 univalents (Figures 5A and 6C). These observations indicated that SCC3, as a member of cohesin complex, had the function of inhibiting inter-sister chromatids exchange during meiosis. We also observed chromosome behaviors of rec8 and rec8 scc3 double mutants from diakinesis to anaphase I (Figure 6C). Both of these mutants contained defective chromosomes displaying a highly sticky phenotype from diakinesis to metaphase I, which is similar to the phenotype of non-homolog end joining (NHEJ). Moreover, chromosome fragmentation appeared in anaphase I, illustrating that meiotic DSBs were not well-repaired in rec8 and rec8 scc3. In addition, DMC1 is also responsible for inter-sister exchange during meiosis in both arabidopsis and rice(Kurzbauer et al., 2012; Wang et al., 2016). To test whether DMC1 affects inter-sister repair process in scc3, we generated dmc1 scc3 double mutant. The meiotic defects of dmc1 and dmc1 scc3 were identical from diakinesis to metaphase I, leading to 24 univalents (Figure 6C). Compared with dmc1, the sister chromatids of dmc1 scc3 were completely separated in anaphase I and no chromosome fragmentation were observed. Taken together, meiotic DSBs in scc3 is probably repaired by using sister chromatid as templates, and the inter-sister chromatids exchange in scc3 is independent of DMC1.

The loading of SCC3 onto meiotic chromosomes depends on REC8

After observing that SCC3 is an axial element in meiosis, we evaluated whether its localization can be affected by other meiotic cohesin protein, specially REC8. Firstly, we investigated the number of γH2AX foci to explore the DSB formation in rec8, and found that the DSBs formation was normal in rec8 (Figure 7A). In addition, DSBs ends resection factor COM1 (n=25 for 3 rec8 mutants) and inter-homologue strand-invasion factor RAD51 (n=26 for 3 rec8 mutants) fail to localize in rec8 (Figure 7A). Besides, we detected the distribution of SCC3 in rec8. we found no SCC3 signals in rec8 mutants, suggesting REC8 affects the normal progression of SCC3 loading (n=36 for 3 rec8 mutants). PAIR2 (n=28 for 3 rec8 mutants) could only form short, interrupted stretches and few PAIR3 signals were detectable (n=32 for 3 rec8 mutants). These results suggested that meiotic DSBs formation was normal in rec8, but DSBs ends resection and homologous recombination process were significantly defective. Thus, we speculated that the highly sticky chromosomes and dissociative chromosomes fragmentation from diakinesis to anaphase I in rec8 was caused by COM1-mediated NHEJ pathway.

Figure 7.

PAIR2 and PAIR3 localize on chromosome axes in rice, which is essential for the formation of synaptonemal complex installation. We further investigated the distribution of SCC3 in pair2 and pair3 mutants (Figure 7B). Specifically, SCC3 signals were normal in pair2 and pair3 mutants during meiosis. Moreover, SCC3 distribution was normal in other mutants that affect meiotic DSB formation (spo11-1), strand invasion and homology searching (com1 and dmc1), and synaptonemal complex formation (zep1) (Figure 7B), indicating SCC3 localization is independent of the recombination process.

SCC3 interacts with RAD21.1

The cohesin complex is a ring-shaped protein complex where subunits interact with each other (Li et al., 2018a; Murayama and Uhlmann, 2015; Roig et al., 2014). To confirm this, we performed Y2H assays between several cohesin proteins. Surprisingly, we found SCC3 only interacts with RAD21.1 (Figure 7C), which was further confirmed in BiFC assays (Figure 7D). However, it is worth noting that SCC3 could not interact with other cohesin proteins, including REC8, SMC1 and SMC3 (Figure 7C), indicating that SCC3 could function directly in combination with RAD21.1 in the cohesin complex.

Previous studies showed cohesin regulates genes expression (Cuadrado et al., 2015; Gligoris and Lowe, 2016; Remeseiro et al., 2012), we therefore investigated expression levels of cohesin-related genes in wildtype and scc3 spikelets using RT-qPCR. Interestingly, we found cohesin subunits SCC1 and REC8, as well as centromeric cohesin protector SGO1 were significantly downregulated in scc3 (Figure S10A). In contrast, the cohesin subunits SMC1 and SMC3, as well as lateral/central elements PAIR3 and ZEP1 showed no differences in gene expression. The only upregulated axial element in scc3 spikelets was PAIR2.

We then investigated cohesin gene expression levels in axial/lateral elements mutants (pair2 and pair3) (Figure S10B), and found SMC3, SCC3 and SCC1 were significantly downregulated compared to the wildtype. These findings suggested that SCC3 may thus be involved in the transcriptional regulation of meiosis genes, while axial elements affect cohesin proteins by regulating the expression levels of associated genes.

Discussion

Chromosome segregation during mitosis and meiosis includes a series of highly organized events, such as the formation of a cohesin complex containing different subunits that play multiple functions (e.g., DSB repair, or gene expression regulation) (Moronta-Gines et al., 2019; Nasmyth and Haering, 2009). Here, we show SCC3 is a conserved cohesin subunit affecting vegetative growth and sister chromatid assembly in both mitosis and meiosis. Crucially, our results also indicate SCC3 is a component of LE/AE that is required for homologous pairing and synapsis during meiosis, which is essential for the recombination process and CO formation. More importantly, SCC3, as a member of cohesin complex, may inhibit inter-sister chromatids repair during meiosis, which provides new insights between cohesin and inter-sister/homolog recombination partner choice in meiotic DSBs repair.

Current cohesin complex research mainly focuses on mitosis because the morphology of sister chromatids during meiosis is difficult to observe. In human cells, sister chromatids separate in mitosis in the absence of the SCC3 homologues SA1 and SA2. The former is required for telomere and arm cohesin, whereas the latter is essential for centromere cohesin (Canudas and Smith, 2009). In rice, SCC3 has only a single copy, therefore we observed these all kinds of defects in scc3. SCC3 is not only essential for the stability of sister chromatids cohesin but also plays a role in loading cohesin onto chromosomes. In yeast scc3, the cohesin trimer composed of SCC1, SMC1 and SMC3 remains intact. However, the trimer fails to localize to chromosomes, probably because SCC3 helps maintaining the stability of cohesin (Orgil et al., 2015; Pathania et al., 2021). Additionally, SCC3 contains a conserved DNA binding domain in the SCC3-SCC1 subcomplex, which is required for cohesin association to chromatin (Higashi et al., 2020). In yeast, the SCC3-SCC1 DNA-binding region recruits cohesin complexes to chromosomes and facilitates the function of cohesin during mitotic cell division (Kulemzina et al., 2012; Li et al., 2018a). These studies implied that SCC3-SCC1 interaction is necessary for cohesin ring stability and for maintaining its association with DNA. In Arabidopsis, SCC3 is also involved in vegetative growth of plants, but its role in mitosis remains unclear (Chelysheva et al., 2005). Building from these previous observations, our study demonstrated that SCC3 is encoded by a single gene that participates in early cohesin formation in mitosis. In scc3, vegetative growth was severely inhibited. We found that the replication process of sister chromatids during interphase was abnormal in scc3, possibly because SCC3 is required for cohesin to stably bind to the chromosomes. Also, we observed an increased distance between sister chromatids in prometaphase, but sister chromatids remain partially dissociated not a complete separation, indicating other cohesin subunits are still functional. Taken together, our results suggest SCC3 is a conserved cohesin protein that plays a very important role in mitosis.

SCC3 is also involved in meiotic chromosome structure and plays a crucial role in homologous recombination and inter-sister chromatid repair during meiosis. In vertebrates, SCC3 is encoded by two genes paralogues, including the meiosis-specific subunit STAG3. In human stag3, SIM analysis showed the chromosome axes are regionally separated and the synaptonemal complex is largely reduced (Biswas et al., 2016; Fukuda et al., 2014; Hopkins et al., 2014; Llano et al., 2012). These observations suggest STAG3 is required for synapsis between homologous chromosomes. Compared to vertebrate cells, the morphology of meiocytes is more readily observed during plant meiosis. In Arabidopsis scc3, a total loss of fertility is associated with SCC3’s role in meiosis, with unstable homologous pairing at pachytene. SCC3 is also necessary for monopolar orientation of kinetochores at meiosis I and the maintenance of centromeric cohesin at anaphase I (Chelysheva et al., 2005). In other cohesin mutants, Atscc2 is required for loading of the meiosis-specific cohesin subunit REC8 (Wang et al., 2020), suggesting defects in cohesin-related genes might impact the ability of the cohesin complex to bind chromosomes. Accordingly, in meiosis, we found rice SCC3 has functions in the maintenance of replication process of sister chromatids in preleptotene. SCC3 localizes between homologous chromosomes as an important axial element during meiosis, ensuring correct homologous pairing and synapsis. Meanwhile, SCC3 is required for normal localization of DMC1, RAD51, ZIP4 and HEI10, which is essential for recombination progress and CO formation. Our working model proposes SCC3 is a part of the cohesin ring, and functions in specific association with SCC1 (Figures 8A). Considering SCC3’s role in cohesin and multiple meiotic functions, we speculate that SCC3 may act as LE to stabilize loop boundaries to promote sister chromatids cohesin during meiosis (Figure 8B). More importantly, meiotic homologous pairing and synapsis are defective in scc3, and 24 univalents could be observed at diakinesis and sister chromatids are completely separated at anaphase I with no chromosome fragmentation, which illustrates that meiotic DSBs are well repaired and could only use sister chromatids as a template to repair. Known meiotic components (COM1, DMC1 and RAD51) can be present in scc3 but apparently making no functional contribution to homologous recombination, but these proteins could also have the effect of creating a leading end that can search for an available inter-sister partner, since DMC1 and RAD51 are also involved in inter-sister chromatids repair process. We propose that, when cohesin is present, meiotic recombination exhibits inter-homolog bias to repair. Nonetheless, when cohesin is absent and homologous pairing is defective, recombination exhibits inter-sister bias to repair (Figure 8C). These observations imply that SCC3 acts as a component of cohesin complex, which could block inter-sister chromatids exchange during meiosis.

Figure 8.

Cohesin is a multi-protein complex that plays a vital role establishing sister chromatid cohesin and high-order chromosome architecture in somatic and germ cells. However, distinct meiosis-specific subunits likely perform different functions (Cuadrado et al., 2015; Ishiguro, 2019; Schvarzstein et al., 2010). Our observations suggest that SCC3 and REC8 may play different roles during mitosis and meiosis. REC8 was considered a meiosis-specific protein, which played the role of SCC1 in meiosis to form cohesin complex. In this study, we found REC8 and SCC3 localization patterns were similar but not identical. Notably, REC8 was not present in mitotic cells. However, we found SCC3 is active in mitosis and meiosis, and may be involved in cohesin loading of the replication process of sister chromatids at preleptotene. It’s worth noting that the mechanism of loading cohesin onto chromosomes in mitosis and meiosis was the same. Meiotic cohesin loaded onto chromosomes during interphase, probably when the sporogenous cell was replicating and acquired meiotic fate. This indicates REC8 is not involved in establishing cohesin at this instance. We also observed SCC3 signals at centromeres from diakinesis to metaphase I, but could not detect any REC8 at metaphase I. These different localization patterns also suggested they may have different functions in meiosis. Previous studies on Arabidopsis rec8 mutants showed sticky chromosomes, and it was difficult to explain the reason of the phenotype (Cai et al., 2003; Lambing et al., 2020). Correspondingly, rice rec8 meiocytes also contained defective chromosomes displaying a highly sticky phenotype from the diakinesis to metaphase I (Shao et al., 2011). Here, we found the chromosomes in scc3 was not sticky as observed in rec8 (Figure 6C). Meanwhile, the majority of meiotic proteins (including SCC3) failed to localize in rec8. This indicates the normal localization of SCC3 and other meiotic proteins depends on REC8, which means REC8 may be a fundamental meiotic protein that is the prerequisite for other meiotic proteins loading. Also, the DSBs end processing and inter-sister/homolog strand-invasion are totally defective in rec8. Thus, we speculated that the sticky chromosomes in rec8 may be caused by the NHEJ repair pathway (Figure 8C). The presented results provide that SCC3 acts as cohesin and stabilizes sister chromatid replication process in both mitosis and meiosis, but REC8 only functions in meiosis. Both proteins are independent of each other in mitosis, but related in meiosis, with REC8 representing a more important player in meiosis.

Material and methods

Meiotic chromosome preparation

Young rice panicles were fixed in Carnoy’s solution (ethanol: acetic acid, 3: 1, v/v). Microsporocytes at different meiotic stages were squashed in an acetocarmine solution on glass slides. Moreover, the slides were covered with a coverslip and washed with 45% acetic acid from each side. Subsequently, the slides were frozen in liquid nitrogen for 10 seconds, and then the coverslip was removed as soon as possible ensuring sufficient cells were fixed on the slide. After ethanol gradient dehydration (70%, 90% and 100%), the slides with chromosome spreads were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA) solution. Chromosome behaviors were observed and captured using a ZEISS A2 microscope imaging system with a micro-charge-coupled (micro CCD) device camera.

Antibody production

To generate the antibody against SCC3, a 648 bp C-terminal fragment of SCC3 complementary DNA (amino acids 900-1116) was amplified using primers SCC3-Ab-F and SCC3-Ab-R (Table S1). The PCR product was cloned into the expression vector pET-30a (Novagen, Madison, WI, USA). The fusion peptide expression and purification were carried out as described previously (Miao et al., 2021). The polyclonal antibody was raised from mouse and rabbit. Other antibodies were generated in our laboratory previously.

Immunofluorescence assay

Fresh young panicles were fixed in 4% (w/v) paraformaldehyde for 30 min at room temperature. Different meiotic stages of anthers were squashed in a drop of 1×PBS solution on glass slides. After freezing in liquid nitrogen and ethanol dehydration, slides were incubated with different combinations of diluted antibodies (1:100) in a humid chamber at 37 °C for 2 h. Thereafter, the slides were washed with 1×PBS solution three times and were further incubated for 1 h with the appropriate fluorochrome-coupled secondary antibody, including fluorescein-isothiocyanate-conjugated goat anti-mouse antibody (Southern Biotech, Birmingham, AL, USA), rhodamine-conjugated goat anti-rabbit antibody (Southern Biotech), and AMCA-conjugated goat anti-guinea pig antibody (Jackson ImmunoResearch, West Grove, PA, USA). Slides were washed with 1×PBS solution three times and eventually stained with DAPI. The immunofluorescence signals were analyzed and captured using a ZEISS A2 microscope imaging system.

Fluorescence in situ hybridization

Young panicles of both wild-type and mutants were fixated in Carnoy’s solution (ethanol: acetic acid, 3: 1, v/v). The fluorescence in situ hybridization (FISH) analysis was performed as a detailed protocol previously (Cheng, 2013). The pAtT4 clone containing telomere repeats, the pTa794 clone containing 5S ribosomal RNA genes, and the bulked oligonucleotide probes specific to chromosome 12 were used as probes in FISH analysis. Rhodamine anti-digoxigenin was used for digoxigenin-labeled probes. Chromosome images were captured using an Olympus (Shinjuku-ku, Tokyo, Japan) BX51 fluorescence microscope with a micro CCD camera using software IPLAB4.0 (BD Biosciences, San Jose, CA, USA).

Yeast two-hybrid assay

The yeast two-hybrid (Y2H) assays were conducted by the full-length CDSs of RAD21.1, SCC3, REC8, SMC1 and SMC3 independently cloned into the pGBKT7 or pGADT7 vector. Co-transformants were selected on SD/DDO (SD-Leu-Trp) medium at 30 °C for 3-4 days. Positive interactions of transformants were selected on SD/QDO (SD-Leu-Trp-His-Ade) medium containing aureobasidin A. All primers used to construct plasmids are listed in Table S1. The detailed protocol was described in the manufacturer’s handbook (Yeast protocols handbook; PT3024-1; Clontech).

BiFC assay

To conduct BiFC assays, SCC3 and RAD21.1 were amplified by KOD-plus polymerase and ligated into BiFC vectors, including pSCYNE (SCN) and pSCYCE (SCC). The constructed plasmids were transformed into protoplasts extracted from the young stem of rice etiolated seedlings. After incubation in the dark for 18 h at 28°C, the CFP signals were captured under a confocal laser scanning microscope at an excitation wavelength 405 nm (Leica TCS SP5, Wetzlar, Germany).

Super-resolution structured illumination microscopy and image analysis

Immunofluorescence assays were conducted by the procedures described above. Meiotic cells with high-quality immunofluorescence signals were marked for recording approximate X-Y positions using the ZEISS A2 microscope. Then these cells were repositioned under the DeltaVision microscope (OMX^TM V4; GEhealthcare, Chicago, IL, USA) and the super-resolution images were processed with SoftWoRx (Applied Precision, Issaquah, WA, USA). And the distance between axial elements was measured using _IMAGEJ 1.37a software.

RNA extraction and real-time PCR

Real-time PCR assays were used to detect the expression patterns of SCC3. Total RNA was extracted from different tissues in wild-type including root, stem, leaf, sheath, spikelet, 2 cm long panicle, 2-3 cm long panicle, 3-5 cm long panicle, 5-7 cm long panicle and 7 cm long panicle using TRIzol reagent. After digestion with RNase-free DNaseI to remove genomic DNA, reverse transcription PCR was performed to synthesize cDNA using SuperScript^TM III Reverse Transcriptase by manufacturer’s protocol (Invitrogen). Quantitative real-time PCR assays were performed on the Bio-Rad CFX96 instrument using Evagreen (Biotium, Freemont, CA, USA) with a procedure of 98 °C 3min, 40 cycles of 98 °C 15s and 60 °C 20s. The UBIQUITIN gene was used as an internal reference. Three biological repeats were performed for each sample. Primers used for real-time PCR assays were listed in Table S1.

Phylogenetic analysis

The full-length amino acid sequence of SCC3 was selected as a template and searched by the NCBI blastp tool to identify similar proteins among species. A detailed protocol for further analysis has been reported previously (Li et al., 2018b; Ren et al., 2018; Ren et al., 2020). Filtered sequences were downloaded and used for constructing neighbor-joining trees by M_EGA5 software and processed with EvolvieW (https://evolgenius.info/evolview/).

Multiple sequence alignments

Multiple alignments were performed using the online toolkit M_AFFT (https://toolkit.tuebingen.mpg.de/mafft) and processed with ESP_RIPT3 (http://espript.ibcp.fr/ESPript/ESPript/) by previously detailed reported (Zhao et al., 2021).

Experimental model and subject details

The rice scc3 mutant was produced by CRISPR-Cas9 toolkit in japonica rice variety Yandao 8. Primers used for CRISPR-Cas9 are listed in Table S1. Other meiosis mutants pair3 (Wuxiangjing 9), pair2 (Nipponbare), zep1 (Nipponbare), rec8 (Yandao 8), sgo1 (Yandao 8), spo11-1 (Guangluai 4), dmc1 (Nipponbare) have been previously described (Wang et al., 2016). Yandao 8 was used as the wild-type. All the rice plants were grown in a paddy field under natural rice-growing conditions.

Quantification and statistical analysis

At least three independent biological repeats were performed for each experiment. Values in Figures are means ± SD. The significant difference between the wild type and corresponding mutants was evaluated by two-tailed Student’s t-test and p < 0.05 was considered as a criterion for judging statistically significant difference. The significance labeled in the Figures represents the following meanings: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Acknowledgements

This work was supported by grants the National Natural Science Foundation of China (U2102219, 31930018 and 32201779).

Additional information

Author contributions

Z.C. and Y.Z. conceived the original screening and research plans; Y.Z., L.R., T.Z., H.Y., Y.M., L.C. and Y.S. performed most of the experiments; Y.Z., D.T. and Y.L. designed the experiments and analyzed the data; Y.Z. wrote the article; Z.C. supervised and complemented the writing.

Declaration of interests

The authors declare no competing interests.

Supplemental item

Figure S1 (related to Figure 1). Multiple sequence alignment of SCC3 with its homologs from three different species.

Figure S2 (related to Figure 1). Protein structure and phylogenetic tree analysis of SCC3.

Figure S3 (related to Figure 1). Multiple sequence alignment of SCC3 with its homologs from different species analyzed in evolutionary tree.

Figure S4 (related to Figure 1). Schematic representation of SCC3’s mutation site.

Figure S5 (related to Figure 1). The expression pattern of SCC3 and plant phenotypic statistics of wildtype and scc3 mutants.

Figure S6 (related to Figures 2 and 3). Immunolocalization of SCC3 and REC8 in scc3 meiosis and mitosis.

Figure S7 (related to Figure 3). Immunolocalization of CENH3 and REC8 in wildtype from diakinesis to metaphase I.

Figure S8 (related to Figure 5). Chromosome behaviors of spo11 and cytogenetic analysis of homologous pairing in wildtype and scc3.

Figure S9 (related to Figure 5). Immunolocalization of ZEP1, PAIR2 and PAIR3 in wildtype at zygotene.

Figure S10 (related to Figure 8). The expression levels of cohesin-related genes in wildtype and other mutants.

Table S1. List of primers used in this study.