The Arabidopsis homolog of Scc4/MAU2 is essential for embryogenesis

Abstract

Factors regulating dynamics of chromatin structure have direct impact on expression of genetic information. Cohesin is a multi-subunit protein complex that is crucial for pairing sister chromatids during cell division, DNA repair and regulation of gene transcription and silencing. In non-plant species, cohesin is loaded on chromatin by the Scc2-Scc4 complex (also known as the NIBPL-MAU2 complex). Here, we identify the Arabidopsis homolog of Scc4, which we denote Arabidopsis thaliana (At)SCC4, and show that it forms a functional complex with AtSCC2, the homolog of Scc2. We demonstrate that AtSCC2 and AtSCC4 act in the same pathway, and that both proteins are indispensable for cell fate determination during early stages of embryo development. Mutant embryos lacking either of these proteins develop only up to the globular stage, and show the suspensor overproliferation phenotype preceded by ectopic auxin maxima distribution. We further establish a new assay to reveal the AtSCC4-dependent dynamics of cohesin loading on chromatin in vivo. Our findings define the Scc2-Scc4 complex as an evolutionary conserved machinery controlling cohesin loading and chromatin structure maintenance, and provide new insight into the plant-specific role of this complex in controlling cell fate during embryogenesis.

Phase transitions of a SEC14-like condensate at Arabidopsis plasma membranes regulate root growth

“…Our results indicated a potential cleavage site right after the arginine R 84 , which coincided with the reported in non-plants ESP cleavage φEXXR consensus sequence I 80 EDVR 84 D (Hellmuth and Stemmann, 2020; Liu et al, 2017) which is also conserved in other SFH8-like proteins ( Figure 4D , E, File S3 ). We further established an in vitro ESP cleavage assay in which we confirmed that immunopurified ESP, mitotically activated through co-expression with cyclinD (Xu et al, 2020), could cleave recombinant GST-SFH8 at R 84 ; we confirmed the specificity of our assay by showing the cleavage of the well-known target of ESP in non-plants, the cohesin ( Figure S4A-D ) (Minina et al, 2017).…”

“…Fusion proteins were detected with α-Myc monoclonal antibodies (Roche, Stockholm, Sweden). The following constructs were used: pBKGT7-AtESP domain A (DomA; Moschou et al, 2016); pBKGT7-AtESP Domain C (DomC; Moschou et al, 2016); pBKGT7-AtMau2 (Minina et al, 2017); pBKGT7-cKin7.3; pBKGT7-cKin7.3-motor; pBKGT7-cKin7.3-tail (Moschou et al, 2016); The absence of self-activation was verified by a transformation of the baits alone to select on minimal medium (SD) lacking the amino acids leucine, histidine, and adenine. The baits were transformed into the strain Y2HGold and mated with the Universal Arabidopsis cDNA Library (Clontech) in Y187.…”

“…The baits were transformed into the strain Y2HGold and mated with the Universal Arabidopsis cDNA Library (Clontech) in Y187. For pairwise Y2H assays, the Gateway-compatible pGADT7 vector (Maier et al, 2008) and the yeast Y187 were used, including the following constructs: pGADKT7-SCC2 (Minina et al, 2017); pGADKT7-cKin7.3-tail; pGADKT7-cSFH8; pGADKT7-cSFH8-SEC14 domain (SD); pGADKT7-cSFH8-Nodulin; pGADKT7-cSFH8-Nodulin 2KtoA ; pGADKT7-cSFH8-Nodulin 6Kto A (or other indicated variants).…”

“…In anaphase when the protease separase cleaves the Scc1/ Mcd1 subunit of cohesin (or its Rec8 counterpart in meiotic cells), thereby allowing the sisters to be pulled apart by the mitotic spindle. The Arabidopsis genome encodes four kleisin subunits: the meiosis-specific ones SYN1 and SYN3, involved in gene expression of meiotic genes, but also expressed in somatic cells, and SYN2 and SYN4, which have been suggested to participate in mitotic cell division (Minina et al, 2017). As we have shown previously, plants expressing tagged SYN proteins, except the mitotic cohesin SYN4, were highly susceptible to even mild environmental changes and showed decreased fertility (Minina et al, 2017).…”