The BTB ubiquitin ligases ETO1, EOL1 and EOL2 act collectively to regulate ethylene biosynthesis in Arabidopsis by controlling type-2 ACC synthase levels

Ethylene biosynthesis is directed by a family of 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACS) that convert S-adenosyl-l-methionine to the immediate precursor ACC. Members of the type-2 ACS subfamily are strongly regulated by proteolysis with various signals stabilizing the proteins to increase ethylene production. In Arabidopsis, this turnover is mediated by the ubiquitin/26 S proteasome system, using a broad complex/tramtrack/bric-a-brac (BTB) E3 assembled with the ETHYLENE OVERPRODUCER 1 (ETO1) BTB protein for target recognition. Here, we show that two Arabidopsis BTB proteins closely related to ETO1, designated ETO1-like (EOL1) and EOL2, also negatively regulate ethylene synthesis via their ability to target ACSs for breakdown. Like ETO1, EOL1 interacts with type-2 ACSs (ACS4, ACS5 and ACS9), but not with type-1 or type-3 ACSs, or with type-2 ACS mutants that stabilize the corresponding proteins in planta. Whereas single and double mutants affecting EOL1 and EOL2 do not show an ethylene-related phenotype, they exaggerate the effects caused by inactivation of ETO1, and further increase ethylene production and the accumulation of ACS5 in eto1 plants. The triple eto1 eol1 eol2 mutant phenotype can be effectively rescued by the ACS inhibitor aminoethoxyvinylglycine, and by silver, which antagonizes ethylene perception. Together with hypocotyl growth assays showing that the sensitivity and response kinetics to ethylene are normal, it appears that ethylene synthesis, but not signaling, is compromised in the triple mutant. Collectively, the data indicate that the Arabidopsis BTB E3s assembled with ETO1, EOL1 and EOL2 work together to negatively regulate ethylene synthesis by directing the degradation of type-2 ACS proteins

Creation and Characterization of LRB (Light-Response BTB) IPIF (Phytochrome-Interacting Factor) Mutant Lines in Arabidopsis thaliana

Color poster with text, graphs, charts, and images.In order to better understand how the LRB and PIF genes interact we are taking a genetic approach, creating plants with T (transfer)-DNA disruptions of both LRB and PIF genes. Study of the phenotypes of these plants may shed light on how these two families of genes work together to regulate light responses or plant growth and development in general.National Science Foundation-Research in Undergraduate Institutions (RUI Grants #1354438); Unversity of Wisconsin--Eau Claire Differential Tuition; University of Wisconsin--Eau Claire Office of Research and Sponsored Programs

Analysis of the PHYB Gene Sequence in Arabidopsis Lines Identified in a Red-Light Genetic Suppressor Screen

Color poster with text, images, and graphs.The capability to detect the amount and quality of light is critical for plant growth and development. Red and far-red light are detected by the phytochrome (phy) photoreceptors which mediate plant behavior1. The flowering plant Arabidopsis thaliana contains the genes LRB1 and LRB2 (Light-Response BTB 1 and 2) which encode proteins functioning as target adaptors in BTB/Cullin 3 E3 ubiquitin-ligase complexes. These complexes target phytochromes for ubiquitylation and degradation2. The phytochromes are red/far-red light receptors and plants containing mutations of both LRB1 and 2 genes express hypersensitivity to red light due to increases levels of these photoreceptors. In an attempt to identify additional genes involved in red-light response, the Gingerich lab conducted a genetic screen to identify mutations which suppress the phenotype caused by the lrb1 lrb2 double mutations. All suppressor mutants thus far analyzed have had mutations in the PHYB gene, which encodes a phytochrome that functions as the major red-light receptor in Arabidopsis. Over the past year we have sequenced parts of the PHYB gene in 6 suppressor mutants that had not yet been analyzed. Mutations in PHYB are potentially interesting for researchers who study this important mediator of plant environmental responses. Alternatively, absence of a mutation within the PHYB gene in these lines could indicate a mutation in a separate gene involved in red light response, which could lead to the discovery of new genes involved in the response.National Science Foundation-Research in Undergraduate Institutions (RUI) grants (#0919678 and #1354438); National Science Foundation Arabidopsis 2010 Program Grant (MCB-0115870); National Institutes of Health Ruth L. Kirschstein Postdoctoral Fellowship (F32-GM68361); University of Wisconsin--Eau Claire Office of Research and Sponsored Program

Analyses of E3 Ubiquitin-Ligase Target Adapter-Encoding BTB Gene Families in Viridiplantae

Color poster with text, figures and tables.Ubiquitylation, the covalent attachment of the small protein ubiquitin (Ub), can modify the degradation, localization, or activity of target proteins and is crucial for proper organism function. This attachment is achieved by the sequential action of E1(Ub-activating), E2 (Ub-conjugating), and E3 (Ub-ligase) enzymes. The E3 complexes bind the target protein and facilitate attachment of the Ub moiety. There are multiple families of E3’s encoded in eukaryotic genomes; one of these are the CRL3 type. CRL3 E3 complexes consist of three proteins; a BTB (Bric-a-Brac, Tramtrack, Broad Complex) domain-containing protein, Cullin 3, and RBX1. The BTB proteins are the target-adapters, binding to the proteins to be ubiquitylated via motifs appended to the ~100 amino-acid BTB domain. Genes encoding BTB proteins have been identified in a wide range of eukaryotic organisms (including fungi, protists, animals, and plants) but the BTB gene families in different groups show great variability in size, complexity, and composition. In land plant genomes thus far studied, BTB gene families are large (~75-150 members) and complicated (with multiple subtypes based on the presence of a diverse set of target-binding motifs). We are interested in when the particular CRL3 family composition seen in the higher plants (as defined by the BTB target-adapter repertoire available) may have arisen in evolution.National Science Foundation-Research in Undergraduate Institutions (RUI) grant (#1354438); University of Wisconsin--Eau Claire Office of Research and Sponsored Program

Can These Mutations Lead to Blindness? : Analyzing the Effects of Genetic Variants of Uncertain Significance

Color poster with text, charts, and images.Genetic testing involves examining a patient's DNA sequence to look for changes (mutations) in the DNA that can potentially cause disease or illness. While some of the changes are benign, many have not yet been characterized and are thus classified as variants of uncertain significance (VUS). In collaboration with Prevention Genetics, our lab has begun to analyze VUS that are predicted to affect the splicing of genes related to ocular diseases. Disruption or alteration of splicing can affect gene function and lead to disease. We are using a minigene system in which the gene segment under investigation is cloned into a plasmid vector and then transfected into eukaryotic cell culture. The mRNA transcripts are then collected and analyzed to determine the effects of the variant on the transcript. Analysis of VUS could lead to a change in variants’ interpretation and directly have an impact on patients and their families by supporting diagnosis and access to treatment.University of Wisconsin--Eau Claire Office of Research and Sponsored Program

Creation and Characterization of LRB (Light-Response BTB) / PIF (Phytochrome-Interacting Factor) Mutant Lines in Arabidopsis thaliana

Color poster with text and images.Light is vital to plant survival and thus plants have developed sophisticated pathways to respond properly to their light environments. Plants sense specific wavelengths of light via photoreceptors, one family of which are the red (R)/far-red (FR)-absorbing phytochromes (phys). Absorption of red light activates the phys, which causes their translocation from the cytosol to the nucleus where they modulate gene expression. They do so by regulating the activity and levels of a family of transcription factors called Phytochrome-Interacting Factors (PIFs). In response to red light, the active phys cause PIFs to be ubiquitylated and degraded, which activates expression of PIF-repressed genes. There is feedback regulation of this pathway as, in response to red light; the PIFs also induce ubiquitylation and degradation of the phys. Work by our lab and others has implicated two genes (called Light-Response BTB 1 and 2 [LRB1 and LRB2]) as critical regulators of the phy/PIF light-response pathway. LRB1 and LRB2 encode BTB (Bric-a-Brac, Tramtrack, Broad Complex) domain-containing proteins that act as target adapters in E3 ubiquitin-ligase complexes. Plants with disruptions of the LRB genes have reduced light-dependent degradation of phys and, like plants with disruptions of PIF genes, exhibit hypersensitivity to red light. The mechanism by which the LRBs modulate phy levels is not entirely clear, however a report published recently showed the LRBs can bind to a complex of a PIF protein (PIF3) and a phy (phyB), leading to ubiquitylation and degradation of both PIF3 and phyB. In order to better understand how the LRB and PIF genes interact we are taking a genetic approach, creating plants with disruptions of both LRB and PIF genes. Study of the phenotypes of these plants may shed light on how these two families of genes work together to regulate red light responses.National Science Foundation-Research in Undergraduate Institutions (RUI) grants (#0919678 and #1354438); National Science Foundation Arabidopsis 2010 Program Grant (MCB-0115870); National Institutes of Health Ruth L. Kirschstein Postdoctoral Fellowship (F32-GM68361); University of Wisconsin--Eau Claire Office of Research and Sponsored Program

Large-Scale, Lineage-Specific Expansion of a Bric-a-Brac/Tramtrack/Broad Complex Ubiquitin-Ligase Gene Family in Rice[W]

Selective ubiquitination of proteins is directed by diverse families of ubiquitin-protein ligases (or E3s) in plants. One important type uses Cullin-3 as a scaffold to assemble multisubunit E3 complexes containing one of a multitude of bric-a-brac/tramtrack/broad complex (BTB) proteins that function as substrate recognition factors. We previously described the 80-member BTB gene superfamily in Arabidopsis thaliana. Here, we describe the complete BTB superfamily in rice (Oryza sativa spp japonica cv Nipponbare) that contains 149 BTB domain–encoding genes and 43 putative pseudogenes. Amino acid sequence comparisons of the rice and Arabidopsis superfamilies revealed a near equal repertoire of putative substrate recognition module types. However, phylogenetic comparisons detected numerous gene duplication and/or loss events since the rice and Arabidopsis BTB lineages split, suggesting possible functional specialization within individual BTB families. In particular, a major expansion and diversification of a subset of BTB proteins containing Meprin and TRAF homology (MATH) substrate recognition sites was evident in rice and other monocots that likely occurred following the monocot/dicot split. The MATH domain of a subset appears to have evolved significantly faster than those in a smaller core subset that predates flowering plants, suggesting that the substrate recognition module in many monocot MATH-BTB E3s are diversifying to ubiquitinate a set of substrates that are themselves rapidly changing. Intriguing possibilities include pathogen proteins attempting to avoid inactivation by the monocot host

Analysis of PHYB Mutations Identified in a Genetic Enhancer Screen in Arabidopsis thaliana

Color poster with text, images, charts, and graphs.Plants and algae use phytochrome (phy) receptors to detect red and far-red light. Changes in phy activity mediate a variety of developmental responses. The model flowering plant Arabidopsis thaliana encodes five phys in its genome, and of these phyB is the primary mediator of red-light responses. In Arabidopsis the Light-Response BTB 1 and 2 (LRB1 and LRB2) proteins function as target adapters in E3 ubiquitin-ligase complexes that target phys for ubiquitylation and degradation. Plants with mutations in both the LRB1 and LRB2genes (lrb1 lrb2 mutants) have increased levels of phyB and display hypersensitivity to red light. To discover additional genes involved in light responses, the Gingerich lab conducted genetic enhancer screens utilizing the lrb1 lrb2 mutants, identifying individuals that exhibited further enhanced red light hypersensitivity. We have mapped and putatively identified the enhancer mutations in two of the lines (E2-1-2 and E11-6-5). In both the putative enhancer mutations are within the PHYB gene.National Science Foundation-Research in Undergraduate Institutions (RUI) grant (#1354438); University of Wisconsin--Eau Claire Office of Research and Sponsored Program

The BTB Ubiqutin Ligases ETO1, EOL1 and EOL2 Act Collectively to Regulate Ethylene Biosynthesis in Arabidopsis by Controlling Type-2 ACC Synthase Levels

Ethylene biosynthesis is directed by a family of 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACS) that convert S-adenosyl-l-methionine to the immediate precursor ACC. Members of the type-2 ACS subfamily are strongly regulated by proteolysis with various signals stabilizing the proteins to increase ethylene production. In Arabidopsis, this turnover is mediated by the ubiquitin/26 S proteasome system, using a broad complex/tramtrack/bric-a-brac (BTB) E3 assembled with the ETHYLENE OVERPRODUCER 1 (ETO1) BTB protein for target recognition. Here, we show that two Arabidopsis BTB proteins closely related to ETO1, designated ETO1-like (EOL1) and EOL2, also negatively regulate ethylene synthesis via their ability to target ACSs for breakdown. Like ETO1, EOL1 interacts with type-2 ACSs (ACS4, ACS5 and ACS9), but not with type-1 or type-3 ACSs, or with type-2 ACS mutants that stabilize the corresponding proteins in planta. Whereas single and double mutants affecting EOL1 and EOL2 do not show an ethylene-related phenotype, they exaggerate the effects caused by inactivation of ETO1, and further increase ethylene production and the accumulation of ACS5 in eto1 plants. The triple eto1 eol1 eol2 mutant phenotype can be effectively rescued by the ACS inhibitor aminoethoxyvinylglycine, and by silver, which antagonizes ethylene perception. Together with hypocotyl growth assays showing that the sensitivity and response kinetics to ethylene are normal, it appears that ethylene synthesis, but not signaling, is compromised in the triple mutant. Collectively, the data indicate that the Arabidopsis BTB E3s assembled with ETO1, EOL1 and EOL2 work together to negatively regulate ethylene synthesis by directing the degradation of type-2 ACS proteins