Summary: CRABS CLAW (CRC) orthologues play a crucial role in floral meristem (FM) determinacy and gynoecium formation across angiosperms, the key developmental processes for ensuring successful plant reproduction and crop production. However, the mechanisms behind CRC mediated FM termination are far from fully understood. Here, we addressed the functional characterization of tomato (Solanum lycopersicum) paralogous CRC genes. Using mapping‐by‐sequencing, RNA interference and CRISPR/Cas9 techniques, expression analyses, protein–protein interaction assays and Arabidopsis complementation experiments, we examined their potential roles in FM determinacy and carpel formation. We revealed that the incomplete penetrance and variable expressivity of the indeterminate carpel‐inside‐carpel phenotype observed in fruit iterative growth (fig) mutant plants are due to the lack of function of the S. lycopersicum CRC homologue SlCRCa. Furthermore, a detailed functional analysis of tomato CRC paralogues, SlCRCa and SlCRCb, allowed us to propose that they operate as positive regulators of FM determinacy by acting in a compensatory and partially redundant manner to safeguard the proper formation of flowers and fruits. Our results uncover for the first time the physical interaction of putative CRC orthologues with members of the chromatin remodelling complex that epigenetically represses WUSCHEL expression through histone deacetylation to ensure the proper termination of floral stem cell activity.

N-α-terminal acetylation is one of the most common, but least understood modifications of eukaryotic proteins. Although a high degree of conservation exists between the N-α-terminal acetylomes of plants and animals, very little information is available on this modification in plants. In yeast and humans, N-α-acetyltransferase complexes include a single catalytic subunit and one or two auxiliary subunits. Here, we report the positional cloning of TRANSCURVATA2 (TCU2), which encodes the auxiliary subunit of the NatB N-α-acetyltransferase complex in Arabidopsis. The phenotypes of loss-of-function tcu2 alleles indicate that NatB complex activity is required for flowering time regulation and for leaf, inflorescence, flower, fruit and embryonic development. In double mutants, tcu2 alleles synergistically interact with alleles of ARGONAUTE10, which encodes a component of the microRNA machinery. In summary, NatB-mediated N-α-terminal acetylation of proteins is pleiotropically required for Arabidopsis development and seems to be functionally related to the microRNA pathway.

Ribosome biogenesis is fundamental to growth and development in eukaryotes and is linked to human diseases and cancer. Arabidopsis thaliana MORPHOLOGY OF ARGONAUTE1-52 SUPPRESSED 2 (MAS2) participates in splicing and 45S ribosomal DNA (rDNA) expression. In a screen for MAS2 interactors, we identified RIBOSOMAL RNA PROCESSING 7 (RRP7), an ortholog of yeast ribosomal RNA processing protein 7 (Rrp7), which is required for 18S ribosomal RNA (rRNA) maturation. Arabidopsis rrp7 mutants exhibit a pleiotropic phenotype including slow growth, altered shoot phyllotaxy, aberrant venation in lateral organs, partial infertility, and abscisic acid hypersensitivity in seedlings. In Arabidopsis, RRP7 localizes mainly to the nucleolus, the site of the 45S rDNA transcription that produces a 45S pre-rRNA primary transcript, precursor of the 25S, 18S and 5.8S rRNAs. Lack of RRP7 function perturbs 18S rRNA maturation, causes nucleolar hypertrophy, and results in an increased 25S/18S rRNA ratio. Arabidopsis contains hundreds of 45S rDNA genes whose expression is epigenetically regulated, and deregulated in rrp7 mutants. Double mutant analysis revealed synergistic interactions between RRP7 alleles and alleles of MAS2, NUCLEOLIN1 (NUC1) and HISTONE DEACETYLASE 6 (HDA6), which encode epigenetic regulators of 45S rDNA transcription. Our results reveal the evolutionarily conserved but divergent roles of RRP7 as a ribosome biogenesis factor.

The selective trafficking of proteins and RNAs through the nuclear envelope regulates nuclear-cytoplasmic segregation of macromolecules and is mediated by nucleopore complexes (NPCs), which consist of about 400 nucleoporins (Nups) of about 30 types. Extensive studies of nucleoporin function in yeast and vertebrates showed that Nups function in nucleocytoplasmic trafficking and other processes. However, limited studies of plant Nups have identified only a few mutations, which cause pleiotropic phenotypes including reduced growth and early flowering. Here, we describe loss-of-function alleles of Arabidopsis TRANSCURVATA1 (TCU1); these mutations cause increased hypocotyl and petiole length, reticulate and asymmetrically epinastic leaf laminae of reduced size, and early flowering. TCU1 is transcribed in all of the organs and tissues examined, and encodes the putative ortholog of yeast and vertebrate Nup58, a nucleoporin of the Nup62 subcomplex. Nup58 forms the central channel of the NPC and acts directly in translocation of proteins through the nuclear envelope in yeast and vertebrates. Yeast two-hybrid (Y2H) assays identified physical interactions between TCU1/NUP58 and 34 proteins, including nucleoporins, SCF (Skp1/Cul1/F-box) ubiquitin ligase complex components and other nucleoplasm proteins. Genetic interactions were also found between TCU1 and genes encoding nucleoporins, soluble nuclear transport receptors and components of the ubiquitin-proteasome and auxin signaling pathways. These genetic and physical interactions indicate that TCU1/NUP58 is a member of the Nup62 subcomplex of the Arabidopsis NPC. Our findings also suggest regulatory roles for TCU1/NUP58 beyond its function in nucleocytoplasmic trafficking, a hypothesis that is supported by the Y2H and genetic interactions that we observed.

Ribosome biogenesis requires stoichiometric amounts of ribosomal proteins and rRNAs. Synthesis of rRNAs consumes most of the transcriptional activity of eukaryotic cells, but its regulation remains largely unclear in plants. We conducted a screen for ethyl methanesulfonate-induced suppressors of Arabidopsis thaliana ago1-52, a hypomorphic allele of AGO1 (ARGONAUTE1), a key gene in microRNA pathways. We identified nine extragenic suppressors as alleles of MAS2 (MORPHOLOGY OF AGO1-52 SUPPRESSED2). Positional cloning showed that MAS2 encodes the putative ortholog of NKAP (NF-κ B activating protein), a conserved eukaryotic protein involved in transcriptional repression and splicing in animals. The mas2 point mutations behave as informational suppressors of ago1 alleles that cause missplicing. MAS2 is a single-copy gene whose insertional alleles are embryonic lethal. In yeast two-hybrid assays, MAS2 interacted with splicing and ribosome biogenesis proteins, and fluorescence in situ hybridization showed that MAS2 colocalizes with the 45S rDNA at the nucleolar organizer regions (NORs). The artificial microRNA amiR-MAS2 partially repressed MAS2 and caused hypomethylation of 45S rDNA promoters as well as partial NOR decondensation, indicating that MAS2 negatively regulates 45S rDNA expression. Our results thus reveal a key player in the regulation of rRNA synthesis in plants.