Molecular and Genetic Analysis of the effects of SUMOylation on the regulation of floral transition in Arabidopsis

SUMOylation, the post-translational attachment of SUMO (Small Ubiquitin-like Modifier) to a substrate protein, regulates the activity of several proteins involved in critical cellular processes like cell division and transcriptional regulation. SUMO is subsequently removed from substrates by SUMO-specific proteases, making this modification reversible. In plants, SUMOylation has been implicated in several physiological responses and flowering time control. ESD4 (Early in Short Days 4) encodes a SUMO-specific protease that prevents the accumulation of SUMO-conjugates in Arabidopsis. The esd4-1 mutant shows a very early flowering phenotype as well as several shoot developmental distortions suggesting an important role of SUMOylation in the regulation of plant development. To investigate the role of SUMOylation in flowering time control a suppressor screen of esd4 was performed. 120 independent suppressors of esd4 (sed) were isolated and 15 of them further characterized. The SUMO-conjugate levels of these seds are more similar to those of esd4-1 than to the wild type. Rough map positions for five of these sed mutants were established using classical genetic methods, and combined with Next Generation Sequencing sed111-1 was finemapped to a region of chromosome I that contains only six candidate genes. In a different study, SUMOylation of SHORT VEGETATIVE PHASE (SVP) was assessed and SUMO attachment lysines were determined using E. coli strains that recapitulate the SUMO conjugation pathway. SVP interacts with FLOWERING LOCUS C (FLC) to form a strong floral repressor complex. To study the role of SUMOylation in SVP function, an svp-null mutant (svp-41) was transformed with constructs aiming to hyperSUMOylate (translational fusions with SUMO or AtSCE) or hypoSUMOylate (mutations in the putative SUMO-attachment sites) the SVP protein in transgenic plants. Mutant phenotypes caused by these constructs are discussed

Arabidopsis thaliana SPF1 and SPF2 are nuclear-located ULP2-like SUMO proteases that act downstream of SIZ1 in plant development

Post-translational modifiers such as the small ubiquitin-like modifier (SUMO) peptide act as fast and reversible protein regulators. Functional characterization of the sumoylation machinery has determined the key regulatory role that SUMO plays in plant development. Unlike components of the SUMO conjugation pathway, SUMO proteases (ULPs) are encoded by a relatively large gene family and are potential sources of specificity within the pathway. This study reports a thorough comparative genomics and phylogenetic characterization of plant ULPs, revealing the presence of one ULP1-like and three ULP2-like SUMO protease subgroups within plant genomes. As representatives of an under-studied subgroup, Arabidopsis SPF1 and SPF2 were subjected to functional characterization. Loss-of-function mutants implicated both proteins with vegetative growth, flowering time, and seed size and yield. Mutants constitutively accumulated SUMO conjugates, and yeast complementation assays associated these proteins with the function of ScUlp2 but not ScUlp1. Fluorescence imaging placed both proteins in the plant cell nucleoplasm. Transcriptomics analysis indicated strong regulatory involvement in secondary metabolism, cell wall remodelling, and nitrate assimilation. Furthermore, developmental defects of the spf1-1 spf2-2 (spf1/2) double-mutant opposed those of the major E3 ligase siz1 mutant and, most significantly, developmental and transcriptomic characterization of the siz1 spf1/2 triple-mutant placed SIZ1 as epistatic to SPF1 and SPF2.We thank Mark Hochstrasser (Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA) for kindly providing the ulp1-ts yeast mutant strain. This research was funded by FEDER (through COMPETE), and by Fundacao para a Ciencia e Tecnologia (FCT), within the scope of project SUMOdulator (FCOMP-01-0124-FEDER-028459 and PTDC/BIA-PLA/3850/2012). PHC was supported by FCT (SFRH/BD/44484/2008). HA and FF were supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000007 and Norte-01-0145-FEDER-000008, respectively). The work was supported by FEDER through the COMPETE 2020-Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT, within the framework of projects 'Rede de Investigacao em Biodiversidade e Biologia Evolutiva' (POCI-01-0145-FEDER-006821) and 'Institute for Research and Innovation in Health Sciences' (POCI-01-0145-FEDER-007274). This research was also supported by a grant from the Spanish Ministerio de Ciencia y Tecnologia (AGL2016-75819-C2-1-R) and FEDER (PCQ, AGC, ERB)

Conservation of the Ustilago maydis effector See1 in related smuts

Ustilago maydis is a biotrophic fungus that induces formation of tumors in maize (Zea mays L). In a recent study we identified See1 (Seedling efficient effector 1) as an U. maydis organ-specific effector required for tumor formation in leaves. See1 is required for U. maydis induced reactivation of plant DNA synthesis during leaf tumor progression. The protein is secreted from biotrophic hyphae and localizes to the cytoplasm and nucleus of plant cell. See1 interacts with maize SGT1, a cell cycle and immune regulator, interfering with its MAPK-triggered phosphorylation. Here, we present new data on the conservation of See1 in other closely related smuts and experimental data on the functionality of See1 ortholog in Ustilago hordei, the causal agent of barley covered smut disease

Identification of Arabidopsis SUMO-interacting proteins that regulate chromatin activity and developmental transitions

Posttranslational modification of proteins by small ubiquitin-like modifier (SUMO) plays essential roles in eukaryotic growth and development. Many covalently modified SUMO targets have been identified; however, the extent and significance of noncovalent interactions of SUMO with cellular proteins is poorly understood. Here, large-scale yeast two-hybrid screens repeatedly identified a surprisingly small number of proteins that interacted with three Arabidopsis SUMO isoforms. These SUMO-interacting proteins are nuclear and fall into two main categories: six histone or DNA methyltransferses or demethylases and six proteins that we show to be the evolutionary and functional homologs of SUMO-targeted ubiquitin ligases (STUbLs). The selectivity of the screen for several methylases and demethylases suggests that SUMO interaction with these proteins has a significant impact on chromatin methylation. Furthermore, the Arabidopsis STUbLs (AT-STUbLs) complemented to varying degrees the growth defects of the Schizosaccharomyces pombe STUbL mutant rfp1/rfp2, and three of them also complemented the genome integrity defects of this mutant, demonstrating that these proteins show STUbL activity. We show that one of the AT-STUbLs least related to the S. pombe protein, AT-STUbL4, has acquired a plant-specific function in the floral transition. It reduces protein levels of CYCLING DOF FACTOR 2, hence increasing transcript levels of CONSTANS and promoting flowering through the photoperiodic pathway

Cell type specific transcriptional reprogramming of maize leaves during Ustilago maydis induced tumor formation

Ustilago maydis is a biotrophic pathogen and well-established genetic model to understand the molecular basis of biotrophic interactions. U. maydis suppresses plant defense and induces tumors on all aerial parts of its host plant maize. In a previous study we found that U. maydis induced leaf tumor formation builds on two major processes: the induction of hypertrophy in the mesophyll and the induction of cell division (hyperplasia) in the bundle sheath. In this study we analyzed the cell-type specific transcriptome of maize leaves 4 days post infection. This analysis allowed identification of key features underlying the hypertrophic and hyperplasic cell identities derived from mesophyll and bundle sheath cells, respectively. We examined the differentially expressed (DE) genes with particular focus on maize cell cycle genes and found that three A-type cyclins, one B-, D- and T-type are upregulated in the hyperplasic tumorous cells, in which the U. maydis effector protein See1 promotes cell division. Additionally, most of the proteins involved in the formation of the pre-replication complex (pre-RC, that assure that each daughter cell receives identic DNA copies), the transcription factors E2F and DPa as well as several D-type cyclins are deregulated in the hypertrophic cells