The protein modifier SUMO is critical for integrity of the Arabidopsis shoot apex at warm ambient temperatures.

SUMO is a protein modification whose conjugate levels peak during acute heat stress. We find that SUMO is also critical for plant longevity when Arabidopsis experiences a prolonged non-damaging period of only 28 degrees Celsius. Remarkably, this thermo-lethality at 28 degrees was not seen with any other mutant of the SUMO pathway tested. Autoimmunity due to low SUMO1/2 expression levels was not causal for this thermo-lethality. The role of SUMO for thermo-resilience was also distinct from its requirement for thermomorphogenesis - a growth response triggered by the same warm temperature, as only the latter response was dependent on the SUMO ligase SIZ1 as well. Thermo-resilience at 28 degrees Celsius and (acquired) thermotolerance (a response that allows plants to recover and acclimate to brief extreme temperatures) both depend on the HEAT SHOCK TRANSCRIPTION FACTOR A1 (HSFA1). Acquired thermotolerance was, however, normal in the sumo1/2 knockdown mutant. Thus, SUMO-dependent thermo-resilience is potentially controlled in a different way than the protein damage pathway that underpins thermotolerance. Close inspection of shoot apices revealed that the cell patterning and tissue integrity of the shoot apex of the SUMO1/2 knockdown mutant was lost at 28, but not 22 degrees Celsius. We thus describe a novel SUMO-dependent phenotype.

The Arabidopsis SUMO E3 ligase SIZ1 mediates the temperature dependent trade-off between plant immunity and growth.

Increased ambient temperature is inhibitory to plant immunity including auto-immunity. SNC1-dependent auto-immunity is, for example, fully suppressed at 28°C. We found that the Arabidopsis sumoylation mutant siz1 displays SNC1-dependent auto-immunity at 22°C but also at 28°C, which was EDS1 dependent at both temperatures. This siz1 auto-immune phenotype provided enhanced resistance to Pseudomonas at both temperatures. Moreover, the rosette size of siz1 recovered only weakly at 28°C, while this temperature fully rescues the growth defects of other SNC1-dependent auto-immune mutants. This thermo-insensitivity of siz1 correlated with a compromised thermosensory growth response, which was independent of the immune regulators PAD4 or SNC1. Our data reveal that this high temperature induced growth response strongly depends on COP1, while SIZ1 controls the amplitude of this growth response. This latter notion is supported by transcriptomics data, i.e. SIZ1 controls the amplitude and timing of high temperature transcriptional changes including a subset of the PIF4/BZR1 gene targets. Combined our data signify that SIZ1 suppresses an SNC1-dependent resistance response at both normal and high temperatures. At the same time, SIZ1 amplifies the dark and high temperature growth response, likely via COP1 and upstream of gene regulation by PIF4 and BRZ1.

Arabidopsis TCP Transcription Factors Interact with the SUMO Conjugating Machinery in Nuclear Foci.

In Arabidopsis more than 400 proteins have been identified as SUMO targets, both in vivo and in vitro. Among others, transcription factors (TFs) are common targets for SUMO conjugation. Here we aimed to exhaustively screen for TFs that interact with the SUMO machinery using an arrayed yeast two-hybrid library containing more than 1,100 TFs. We identified 76 interactors that foremost interact with the SUMO conjugation enzyme SCE1 and/or the SUMO E3 ligase SIZ1. These interactors belong to various TF families, which control a wide range of processes in plant development and stress signaling. Amongst these interactors, the TCP family was overrepresented with several TCPs interacting with different proteins of the SUMO conjugation cycle. For a subset of these TCPs we confirmed that the catalytic site of SCE1 is essential for this interaction. In agreement, TCP1, TCP3, TCP8, TCP14, and TCP15 were readily SUMO modified in an E. coli sumoylation assay. Strikingly, these TCP-SCE1 interactions were found to redistribute these TCPs into nuclear foci/speckles, suggesting that these TCP foci represent sites for SUMO (conjugation) activity.

Genetic characterization of T-DNA insertions in the genome of the Arabidopsis thaliana sumo1/2 knock-down line.

Sumoylation is an essential post-translational modification in Arabidopsis thaliana, which entails the conjugation of the SUMO protein onto lysine residues in target proteins. In Arabidopsis, 2 closely related genes, SUMO1 and SUMO2, act redundantly and are in combination essential for plant development, i.e. the combined loss of SUMO1 and SUMO2 results in embryo-lethality. To circumvent this lethality, SUMO2 was previously knocked down in a sumo1 knockout background by expressing an artificial microRNA that targets SUMO2 (amiR-SUMO2). This sumo1/2KD line with low SUMO2 levels represents a valuable genetics tool to investigate SUMO function in planta. Here, we re-sequenced the whole-genome of this sumo1/2KD line and identified 2 amiR-SUMO2 insertions in this line, which were confirmed by PCR-genotyping. Identification of these 2 insertions enables genetics with this tool.

Whole-genome duplications followed by tandem duplications drive diversification of the protein modifier SUMO in Angiosperms.

The ubiquitin-like modifier (UBL) SUMO (Small Ubiquitin-Like Modifier) regulates protein function. Structural rather than sequence homology typifies UBL families. However, individual UBL types, such as SUMO, show remarkable sequence conservation. Selection pressure also operates at the SUMO gene copy number, as increased SUMO levels activate immunity and alter flowering time in Arabidopsis. We show how, despite this selection pressure, the SUMO family has diversified into eight paralogues in Arabidopsis. Relationships between the paralogues were investigated using genome collinearity and gene tree analysis. We show that palaeopolyploidy followed by tandem duplications allowed expansion and then diversification of the SUMO genes. For example, Arabidopsis SUMO5 evolved from the pan-eudicot palaeohexaploidy event (gamma), which yielded three SUMO copies. Two gamma copies were preserved as archetype SUMOs, suggesting subfunctionalization, whereas the third copy served as a hotspot for SUMO diversification. The Brassicaceae-specific alpha duplication then caused the duplication of one archetype gamma copy, which, by subfunctionalization, allowed the retention of both SUMO1 and SUMO2. The other archetype gamma copy was simultaneously pseudogenized (SUMO4/6). A tandem duplication of SUMO2 subsequently yielded SUMO3 in the Brassicaceae crown group. SUMO3 potentially neofunctionalized in Arabidopsis, but it is lost in many Brassicaceae. Our advanced methodology allows the study of the birth and fixation of other paralogues in plants.