The Arabidopsis Auxin Receptor F-Box Proteins AFB4 and AFB5 Are Required for Response to the Synthetic Auxin Picloram.

The plant hormone auxin is perceived by a family of F-box proteins called the TIR1/AFBs. Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6. Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling by promoting the degradation of the Aux/IAA transcriptional repressors. In this report, we demonstrate that both AFB4 and AFB5 also function as auxin receptors based on in vitro assays. We also provide genetic evidence that AFB4 and AFB5 are targets of the picloram family of auxinic herbicides in addition to indole-3-acetic acid. In contrast to previous studies we find that null afb4 alleles do not exhibit obvious defects in seedling morphology or auxin hypersensitivity. We conclude that AFB4 and AFB5 act in a similar fashion to other members of the family but exhibit a distinct auxin specificity.

Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression.

Intrinsically disordered, highly charged protein sequences act as entropic bristles (EBs), which, when translationally fused to partner proteins, serve as effective solubilizers by creating both a large favorable surface area for water interactions and large excluded volumes around the partner. By extending away from the partner and sweeping out large molecules, EBs can allow the target protein to fold free from interference. Using both naturally occurring and artificial polypeptides, we demonstrate the successful implementation of intrinsically disordered fusions as protein solubilizers. The artificial fusions discussed herein have a low level of sequence complexity and a high net charge but are diversified by means of distinctive amino acid compositions and lengths. Using 6xHis fusions as controls, soluble protein expression enhancements from 65% (EB60A) to 100% (EB250) were observed for a 20-protein portfolio. Additionally, these EBs were able to more effectively solubilize targets compared to frequently used fusions such as maltose-binding protein, glutathione S-transferase, thioredoxin, and N utilization substance A. Finally, although these EBs possess very distinct physiochemical properties, they did not perturb the structure, conformational stability, or function of the green fluorescent protein or the glutathione S-transferase protein. This work thus illustrates the successful de novo design of intrinsically disordered fusions and presents a promising technology and complementary resource for researchers attempting to solubilize recalcitrant proteins.

The AFB4 auxin receptor is a negative regulator of auxin signaling in seedlings.

The plant hormone auxin is perceived by a family of F box proteins called the TIR1/auxin-signaling F box proteins (AFBs). Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6. Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling. In this report, we demonstrate a unique role for the AFB4 clade. Both AFB4 and AFB5 function as auxin receptors based on in vitro assays. However, unlike other members of the family, loss of AFB4 results in a range of growth defects that are consistent with auxin hypersensitivity, including increased hypocotyl and petiole elongation and increased numbers of lateral roots. Indeed, qRT-PCR experiments show that afb4-2 is hypersensitive to indole-3-acetic acid (IAA) in the hypocotyl, indicating that AFB4 is a negative regulator of auxin response. Furthermore, we show that AFB4 has a particularly important role in the response of seedlings to elevated temperature. Finally, we provide evidence that the AFB4 clade is the major target of the picloram family of auxinic herbicides. These results reveal a previously unknown aspect of auxin receptor function.

Metalloproteomics and metal toxicology of α-synuclein.

Significant evidence has been accumulated linking exposure to heavy metals and/or distortion of metal homeostasis with the development of various neurodegenerative diseases. α-Synuclein is known to be involved in pathogenesis of a subset of neurodegenerative diseases collectively known as synucleinopathies. Therefore the interplay between metals, α-synuclein and neurodegeneration has attracted significant attention of researchers. This review discusses some of the aspects of the α-synuclein metalloproteomics and represents the peculiarities and consequences of α-synuclein interaction with various metal ions. Both non-specific and specific binding of this protein to metals is considered together with the analysis of the effects of such interactions on α-synuclein structure and aggregation propensity.

The ubiquitin-proteasome system regulates plant hormone signaling.

Plants utilize the ubiquitin-proteasome system (UPS) to modulate nearly every aspect of growth and development. Ubiquitin is covalently attached to target proteins through the action of three enzymes known as E1, E2, and E3. The ultimate outcome of this post-translational modification depends on the nature of the ubiquitin linkage and the extent of polyubiquitination. In most cases, ubiquitination results in degradation of the target protein in the 26S proteasome. During the last 10 years it has become clear that the UPS plays a prominent regulatory role in hormone biology. E3 ubiquitin ligases in particular actively participate in hormone perception, de-repression of hormone signaling pathways, degradation of hormone specific transcription factors, and regulation of hormone biosynthesis. It is certain that additional functions will be discovered as more of the nearly 1200 potential E3s in plants are elucidated.

Recent advances and emerging trends in plant hormone signalling.

Plant growth and development is regulated by a structurally unrelated collection of small molecules called plant hormones. During the last 15 years the number of known plant hormones has grown from five to at least ten. Furthermore, many of the proteins involved in plant hormone signalling pathways have been identified, including receptors for many of the major hormones. Strikingly, the ubiquitin-proteasome pathway plays a central part in most hormone-signalling pathways. In addition, recent studies confirm that hormone signalling is integrated at several levels during plant growth and development.

Plant hormones are versatile chemical regulators of plant growth.

The plant hormones are a structurally unrelated collection of small molecules derived from various essential metabolic pathways. These compounds are important regulators of plant growth and mediate responses to both biotic and abiotic stresses. During the last ten years there have been many exciting advances in our understanding of plant hormone biology, including new discoveries in the areas of hormone biosynthesis, transport, perception and response. Receptors for many of the major hormones have now been identified, providing new opportunities to study the chemical specificity of hormone signaling. These studies also reveal a surprisingly important role for the ubiquitin-proteasome pathway in hormone signaling. In addition, recent work confirms that hormone signaling interacts at multiple levels during plant growth and development. In the future, a major challenge will be to understand how the information conveyed by these simple compounds is integrated during plant growth.

New auxin analogs with growth-promoting effects in intact plants reveal a chemical strategy to improve hormone delivery.

Plant growth depends on the integration of environmental cues and phytohormone-signaling pathways. During seedling emergence, elongation of the embryonic stem (hypocotyl) serves as a readout for light and hormone-dependent responses. We screened 10,000 chemicals provided exogenously to light-grown seedlings and identified 100 compounds that promote hypocotyl elongation. Notably, one subset of these chemicals shares structural characteristics with the synthetic auxins, 2,4-dichlorophenoxyacetic acid (2,4-D), and 1-naphthaleneacetic acid (1-NAA); however, traditional auxins (e.g., indole-3-acetic acid [IAA], 2,4-D, 1-NAA) have no effect on hypocotyl elongation. We show that the new compounds act as "proauxins" akin to prodrugs. Our data suggest that these compounds diffuse efficiently to the hypocotyls, where they undergo cleavage at varying rates, releasing functional auxins. To investigate this principle, we applied a masking strategy and designed a pro-2,4-D. Unlike 2,4-D alone, this pro-2,4-D enhanced hypocotyl elongation. We further demonstrated the utility of the proauxins by characterizing auxin responses in light-grown hypocotyls of several auxin receptor mutants. These new compounds thus provide experimental access to a tissue previously inaccessible to exogenous application of auxins. Our studies exemplify the combined power of chemical genetics and biochemical analyses for discovering and refining prohormone analogs with selective activity in specific plant tissues. In addition to the utility of these compounds for addressing questions related to auxin and light-signaling interactions, one can envision using these simple principles to study other plant hormone and small molecule responses in temporally and spatially controlled ways.

Degradation of the cyclin-dependent kinase inhibitor KRP1 is regulated by two different ubiquitin E3 ligases.

In animals and fungi, a group of proteins called the cyclin-dependent kinase inhibitors play a key role in cell cycle regulation. However, comparatively little is known about the role of these proteins in plant cell cycle regulation. To gain insight into the mechanisms by which the plant cell cycle is regulated, we studied the cyclin-dependent kinase inhibitor KRP1 in Arabidopsis. KRP1 interacts with the CDKA;1/CYCD2;1 complex in planta and functions in the G1-S transition of the cell cycle. Furthermore, we show that KRP1 is a likely target of the ubiquitin/proteasome pathway. Two different ubiquitin protein ligases, SCF(SKP2) and the RING protein RKP, contribute to its degradation. These results suggest that SCF(SKP2b) and RPK play an important role in the cell cycle through regulating KRP1 protein turnover.

The WAG1 and WAG2 protein kinases negatively regulate root waving in Arabidopsis.

The WAG1 and WAG2 genes of Arabidopsis thaliana encode protein-serine/threonine kinases that are closely related to PINOID. In order to determine what roles WAG1 and WAG2 play in seedling development, we used a reverse genetics approach to study the wag1, wag2 and wag1/wag2 mutant phenotypes for clues. Although the wag mutants do not contain detectable amounts of the corresponding mRNA, they are wild type in most respects. However, wag1/wag2 double mutants exhibit a pronounced wavy root phenotype when grown vertically on agar plates, a phenotype observed in wild-type plants only on plates inclined to angles less than 90 degrees. The wag1 and wag2 mutants also demonstrate enhanced root waving, but to a lesser extent. Moreover, the double mutant roots are more resistant to the effects of N-1-naphthylphthalamic acid on the inhibition of root curling, raising the possibility that transport of auxin is affected in the wag mutants. Promoter fusions to the gusA reporter gene demonstrate that the WAG promoters are most active in root tips, consistent with the observed phenotypes in the wag mutants.