Age-dependent leaf senescence and cell death in Arabidopsis (Arabidopsis thaliana) requires activation of the transcription factor ORESARA1 (ORE1) and is not initiated prior to a leaf age of 28 d. Here, we investigate the conditional execution of events that regulate early senescence and cell death in senescence-associated ubiquitin ligase1 (saul1) mutants, deficient in the PLANT U-BOX-ARMADILLO E3 ubiquitin ligase SAUL1. In saul1 mutants challenged with low light, the switch of age-dependent cell death was turned on prematurely, as indicated by the accumulation of ORE1 transcripts, induction of the senescence marker gene SENESCENCE-ASSOCIATED GENE12, and cell death. However, ORE1 accumulation by itself was not sufficient to cause saul1 phenotypes, as demonstrated by double mutant analysis. Exposure of saul1 mutants to low light for only 24 h did not result in visible symptoms of senescence; however, the senescence-promoting transcription factor genes WRKY53, WRKY6, and NAC-LIKE ACTIVATED BY AP3/PI were up-regulated, indicating that senescence in saul1 seedlings was already initiated. To resolve the time course of gene expression, microarray experiments were performed at narrow intervals. Differential expression of the genes involved in salicylic acid and defense mechanisms were the earliest events detected, suggesting a central role for salicylic acid in saul1 senescence and cell death. The salicylic acid content increased in low-light-treated saul1 mutants, and application of exogenous salicylic acid was indeed sufficient to trigger saul1 senescence in permissive light conditions. Double mutant analyses showed that PHYTOALEXIN DEFICIENT4 (PAD4) but not NONEXPRESSER OF PR GENES1 (NPR1) is essential for saul1 phenotypes. Our results indicate that saul1 senescence depends on the PAD4-dependent salicylic acid pathway but does not require NPR1 signaling.

Plants have evolved elaborate mechanisms to regulate pathogen defense. Imbalances in this regulation may result in autoimmune responses that are affecting plant growth and development. In Arabidopsis, SAUL1 encodes a plant U-box ubiquitin ligase and regulates senescence and cell death. Here, we show that saul1-1 plants exhibit characteristics of an autoimmune mutant. A decrease in relative humidity or temperature resulted in reduced growth and systemic lesioning of saul1-1 rosettes. These physiological changes are associated with increased expression of salicylic acid–dependent and pathogenesis-related (PR) genes. Consistently, resistance of saul1-1 plants against Pseudomonas syringae pv. maculicola ES4326, P. syringae pv. tomato DC3000, or Hyaloperonospora arabidopsidis Noco2 was enhanced. Transmission electron microscopy revealed alterations in saul1-1 chloroplast ultrastructure and cell-wall depositions. Confocal analysis on aniline blue–stained leaf sections and cellular universal micro spectrophotometry further showed that these cell-wall depositions contain callose and lignin. To analyze signaling downstream of SAUL1, we performed epistasis analyses between saul1-1 and mutants in the EDS1/PAD4/SAG101 hub. All phenotypes observed in saul1-1 plants at low temperature were dependent on EDS1 and PAD4 but not SAG101. Taken together, SAUL1 negatively regulates immunity upstream of EDS1/PAD4, likely through the degradation of an unknown activator of the pathway.

Post-translational protein modification plays a pivotal role in the regulation and specific turnover of proteins. One of these important modifications is the ubiquitination of target proteins, which can occur at distinct cellular compartments. At the plasma membrane, ubiquitination regulates the internalization and thus trafficking of membrane proteins such as receptors and channels. The Arabidopsis plant U-box armadillo repeat (PUB-ARM) ubiquitin ligase SAUL1 (SENESCENCE-ASSOCIATED UBIQUITIN LIGASE1) is part of the ubiquitination machinery at the plasma membrane. In contrast to most other PUB-ARM proteins, SAUL1 carries additional C-terminal ARM repeats responsible for plasma membrane-association. Here, we demonstrated that the C-terminal ARM repeat domain is also essential and sufficient to mediate plasma membrane-association of the closest Arabidopis paralog AtPUB43. We investigated targeting of PUB-ARM ubiquitin ligases of different plant species to find out whether plasma membrane-association of SAUL1-type PUB-ARM proteins is conserved. Phylogenetic analysis identified orthologs of SAUL1 in these plant species. Intracellular localization of transiently expressed GFP fusion proteins revealed that indeed plasma membrane-association due to additional C-terminal ARM repeats represents a conserved feature of SAUL1-type proteins. Analyses of transgenic Arabidopsis plants overexpressing N-terminally masked or truncated proteins revealed that interfering with the function of SAUL1-type proteins resulted in severe growth defects. Our results suggest an ancient origin of ubiquitination at the plasma membrane in the evolution of land plants.

Plant U-box armadillo repeat (PUB-ARM) ubiquitin (Ub) ligases have important functions in plant defense through the ubiquitination of target proteins. Defense against pathogens involves vesicle trafficking and the formation of extracellular vesicles. The PUB-ARM protein SENESCENCE ASSOCIATED UBIQUITIN E3 LIGASE1 (SAUL1) can form patches at the plasma membrane related to tethering multi-vesicular bodies (MVBs) to the plasma membrane. We uncovered the structure of a full-length plant ubiquitin ligase and the structural requirements of SAUL1, which are crucial for its function in patch formation. We resolved the structure of SAUL1 monomers by small-angle X-ray scattering (SAXS). The SAUL1 model showed that SAUL1 consists of two domains: a domain containing the N-terminal U-box and armadillo (ARM) repeats and the C-terminal ARM repeat domain, which includes a positively charged groove. We showed that all C-terminal ARM repeats are essential for patch formation and that this function requires arginine residue at position 736. By applying SAXS to polydisperse SAUL1 systems, the oligomerization of SAUL1 is detectable, with SAUL1 tetramers being the most prominent oligomers at higher concentrations. The oligomerization domain consists of the N-terminal U-box and some N-terminal ARM repeats. Deleting the U-box resulted in the promotion of the SAUL1 tethering function. Our findings indicate that structural changes in SAUL1 may be fundamental to its function in forming patches at the plasma membrane.

The formation and maintenance of the mitotic spindle during cell division requires several microtubule-interacting motor proteins. Members of the kinesin-5 family play an essential role in the bipolar organization of the spindle. These highly conserved, homotetrameric proteins cross-link anti-parallel microtubules and slide them apart to elongate the spindle during the equal separation of chromosomes. Whereas vertebrate kinesin-5 proteins are well studied, knowledge about the biochemical properties and the function of plant kinesin-5 proteins is still limited. Here, we characterized the properties of AtKRP125b, one of four kinesin-5 proteins in Arabidopsis thaliana. In in vitro motility assays, AtKRP125b displayed the archetypal characteristics of a kinesin-5 protein, a low velocity of about 20 nm·s⁻¹, and a plus end-directed, processive movement. Moreover, AtKRP125b was able to cross-link microtubules and to slide them apart, as required for developing and maintaining the mitotic spindle. In line with such a function, GFP-AtKRP125b fusion proteins were predominantly detected in the nucleus when expressed in Arabidopsis thaliana leaf protoplasts or Nicotiana benthamiana epidermis cells and analyzed by confocal microscopy. However, we also detected GFP signals in the cytoplasm, suggesting additional functions. By generating and analyzing AtKRP125b promoter-reporter lines, we showed that the AtKRP125b promoter was active in the vascular tissue of roots, lateral roots, cotyledons, and true leaves. Remarkably, we could not detect promoter activity in meristematic tissues. Taken together, our biochemical data support a role of AtKRP125b in mitosis, but it may also have additional functions outside the nucleus and during interphase.