Jasmina Kurepa

Genetic Analysis of SUMOylation in Arabidopsis: Conjugation of SUMO1 and SUMO2 to Nuclear Proteins Is Essential

The posttranslational addition of small ubiquitin-like modifiers (SUMOs) to other intracellular proteins has been implicated in a variety of eukaryotic functions, including modifying cytoplasmic signal transduction, nuclear import and subnuclear compartmentalization, DNA repair, and transcription regulation. For plants, in particular, both genetic analyses and the rapid accumulation of SUMO conjugates in response to various adverse environmental conditions suggest that SUMOylation plays a key role in the stress response. Through genetic analyses of various SUMO conjugation mutants, we show here that the SUMO1 and SUMO2 isoforms, in particular, and SUMOylation, in general, are essential for viability in Arabidopsis (Arabidopsis thaliana). Null T-DNA insertion mutants affecting the single genes encoding the SUMO-activating enzyme subunit SAE2 and the SUMO-conjugating enzyme SCE1 are embryonic lethal, with arrest occurring early in embryo development. Whereas the single genes encoding the SUMO1 and SUMO2 isoforms are not essential by themselves, double mutants missing both are also embryonic lethal. Viability can be restored by reintroduction of SUMO1 expression in the homozygous sum1-1 sum2-1 background. Various stresses, like heat shock, dramatically increase the pool of SUMO conjugates in planta. This increase involves SUMO1 and SUMO2 and is mainly driven by the SUMO protein ligase SIZ1, with most of the conjugates accumulating in the nucleus. Taken together, it appears that SIZ1-mediated conjugation of SUMO1 and SUMO2 to other intracellular proteins is essential in Arabidopsis, possibly through stress-induced modification of a potentially diverse pool of nuclear proteins.

Uptake and distribution of ultrasmall anatase TiO2 Alizarin red S nanoconjugates in Arabidopsis thaliana.

While few publications have documented the uptake of nanoparticles in plants, this is the first study describing uptake and distribution of the ultrasmall anatase TiO(2) in the plant model system Arabidopsis. We modified the nanoparticle surface with Alizarin red S and sucrose and demonstrated that nanoconjugates traversed cell walls, entered into plant cells, and accumulated in specific subcellular locations. Optical and X-ray fluorescence microscopy coregistered the nanoconjugates in cell vacuoles and nuclei.

Loss of 26S Proteasome Function Leads to Increased Cell Size and Decreased Cell Number in Arabidopsis Shoot Organs

Although the final size of plant organs is influenced by environmental cues, it is generally accepted that the primary size determinants are intrinsic factors that regulate and coordinate cell proliferation and cell expansion. Here, we show that optimal proteasome function is required to maintain final shoot organ size in Arabidopsis (Arabidopsis thaliana). Loss of function of the subunit regulatory particle AAA ATPase (RPT2a) causes a weak defect in 26S proteasome activity and leads to an enlargement of leaves, stems, flowers, fruits, seeds, and embryos. These size increases are a result of increased cell expansion that compensates for a reduction in cell number. Increased ploidy levels were found in some but not all enlarged organs, indicating that the cell size increases are not caused by a higher nuclear DNA content. Partial loss of function of the regulatory particle non-ATPase (RPN) subunits RPN10 and RPN12a causes a stronger defect in proteasome function and also results in cell enlargement and decreased cell proliferation. However, the increased cell volumes in rpn10-1 and rpn12a-1 mutants translated into the enlargement of only some, but not all, shoot organs. Collectively, these data show that during Arabidopsis shoot development, the maintenance of optimal proteasome activity levels is important for balancing cell expansion with cell proliferation rates.

Proteasome regulation, plant growth and stress tolerance

Plant cells contain a mixture of 26S and 20S proteasomes that mediate ubiquitin dependent and ubiquitin independent proteolysis, respectively. The 26S proteasome contains the 20S proteasome and one or two regulatory particles that are required for ubiquitin dependent degradation. Comparative analyses of Arabidopsis proteasome mutants revealed that a decrease in 26S proteasome biogenesis causes heat shock hypersensitivity and reduced cell division rates that are compensated by increased cell expansion. Loss of 26S proteasome function also leads to an increased 20S proteasome biogenesis, which in turn enhances the cellular capacity to degrade oxidized proteins and thus increases oxidative stress tolerance. These findings suggest the intriguing possibility that 26S and 20S proteasome activities are regulated to control plant development and stress responses. This mini review highlights some of the recent studies on proteasome regulation in plants.

Salt Stress–Induced Disassembly of Arabidopsis Cortical Microtubule Arrays Involves 26S Proteasome–Dependent Degradation of SPIRAL1

This study examines the relationship between salt stress tolerance and dynamic instability of microtubules, revealing an important role for proteasome-dependent regulation of SPR1 in the survival of plants challenged by high salinity. The dynamic instability of cortical microtubules (MTs) (i.e., their ability to rapidly alternate between phases of growth and shrinkage) plays an essential role in plant growth and development. In addition, recent studies have revealed a pivotal role for dynamic instability in the response to salt stress conditions. The salt stress response includes a rapid depolymerization of MTs followed by the formation of a new MT network that is believed to be better suited for surviving high salinity. Although this initial depolymerization response is essential for the adaptation to salt stress, the underlying molecular mechanism has remained largely unknown. Here, we show that the MT-associated protein SPIRAL1 (SPR1) plays a key role in salt stress–induced MT disassembly. SPR1, a microtubule stabilizing protein, is degraded by the 26S proteasome, and its degradation rate is accelerated in response to high salinity. We show that accelerated SPR1 degradation is required for a fast MT disassembly response to salt stress and for salt stress tolerance.

Ultra‐small TiO2 nanoparticles disrupt microtubular networks in Arabidopsis thaliana

In spite of the mounting concerns, current understanding of the extent and mechanisms of phytotoxicity of manufactured nanomaterials remains limited. Here we show that in Arabidopsis thaliana, ultra-small anatase TiO(2) nanoparticles cause reorganization and elimination of microtubules followed by the accelerated and 26S proteasome-dependent degradation of tubulin monomers. Similar to other microtubule-disrupting agents, TiO(2) nanoparticles induce isotropic growth of root cells. Because microtubules are essential for the normal function of all eukaryotic cells, these results reveal a potentially important consequence of environmental pollution by this widely used nanomaterial.

The Arabidopsis 26S Proteasome Subunit RPN1a is Required for Optimal Plant Growth and Stress Responses

The current literature offers contradictory results regarding the role of the proteasome subunit RPN1a in Arabidopsis development. Here we show that plants lacking RPN1a are viable and have increased cell sizes, decreased heat shock tolerance, increased oxidative stress tolerance and other phenotypes characteristic for 26S proteasome subunit mutants. These results strengthen our contention that most of the phenotypes of 26S proteasome mutants in Arabidopsis described to date reflect a general impairment in 26S proteasome function rather than a specific defect of a single subunit, and suggest that the role of the RPN1a subunit during embryogenesis needs to be reconsidered.

Direct isolation of flavonoids from plants using ultra‐small anatase TiO2 nanoparticles

Surface functionalization of nanoparticles has become an important tool for in vivo delivery of bioactive agents to their target sites. Here we describe the reverse strategy, nanoharvesting, in which nanoparticles are used as a tool to isolate bioactive compounds from living cells. Anatase TiO₂ nanoparticles smaller than 20 nm form strong bonds with molecules bearing enediol and especially catechol groups. We show that these nanoparticles enter plant cells, conjugate enediol and catechol group-rich flavonoids in situ, and exit plant cells as flavonoid-nanoparticle conjugates. The source plant tissues remain viable after treatment. As predicted by the surface chemistry of anatase TiO₂ nanoparticles, quercetin-based flavonoids were enriched amongst the nanoharvested flavonoid species. Nanoharvesting eliminates the use of organic solvents, allows spectral identification of the isolated compounds, and opens new avenues for use of nanomaterials for coupled isolation and testing of bioactive properties of plant-synthesized compounds.

To misfold or to lose structure? Detection and degradation of oxidized proteins by the 20S proteasome

Aggregation of proteins damaged by stress is often a causal factor of cell death. To prevent aggregation, eukaryotic cells rapidly degrade damaged proteins by engaging two types of proteasomes. The first type is the 26S proteasome (26SP) which is composed of a cylindrical proteolytic core - the 20S proteasome (20SP) - and one or two regulatory particles (RPs) that interact with ubiquitinated proteins. The second type is the free 20SP which mediates ubiquitin-independent proteolysis. We have recently shown that loss of RP function in Arabidopsis leads to an expected decrease in 26SP-dependent protein degradation and hypersensitivity to stresses that induce protein misfolding. Surprisingly, RP mutants have increased 20SP activity and tolerance to oxidative stress. This finding suggests that misfolded proteins carry one type of degradation signal that steers them to ubiquitination enzymes and the 26SP, while oxidatively damaged proteins carry another that guides them directly to the 20SP for degradation. Here we suggest that protein oxidation induces the formation of unstructured regions that serve as targeting signals for 20SP-dependent proteolysis.

Arabidopsis sensitivity to protein synthesis inhibitors depends on 26S proteasome activity

The 26S proteasome (26SP), the central protease of the ubiquitin-dependent proteolysis pathway, controls the regulated proteolysis of functional proteins and the removal of misfolded and damaged proteins. In Arabidopsis, cellular and stress response phenotypes of a number of mutants with partially impaired 26SP function have been reported. Here, we describe the responses of proteasome mutants to protein synthesis inhibitors. We show that the rpt2a-3, rpn10-1 and rpn12a-1 mutants are hypersensitive to the antibiotic hygromycin B, and tolerant to the translation inhibitor cycloheximide (CHX) and herbicide l-phosphinothricin (PPT). In addition to the novel mechanism for herbicide tolerance, our data suggests that the combination of hygromycin B, CHX and PPT growth-response assays could be used as a facile diagnostic tool to detect altered 26SP function in plant mutants and transgenic lines.