To respond to the environment and regulate growth and development, it is critical for organisms to precisely modulate the activity of proteins in their cells. One mechanism by which this regulation occurs is the covalent attachment of ubiquitin (Ub) and other ubiquitin-like proteins (UBLs) to specific target proteins. These modifications can alter the function, location, or levels of the target. Attachment of the majority of these modifiers occurs by an ATP-dependent E1/E2/E3 conjugation cascade during which a thioester linkage is formed between the Ub/UBL and the E1, followed by a transthiolation reaction where the Ub/UBL is transferred to the E2, then attachment of the Ub/UBL to the target protein via an isopeptide bond. This final step is typically mediated by E3 ligases, which recruit the E2, bind the substrate, and catalyze linkage between the Ub/UBL and the target.

Seeds employ sensory systems that assess various environmental cues over time to maximize the successful transition from embryo to seedling. Here we show that the Arabidopsis F-BOX protein COLD TEMPERATURE-GERMINATING (CTG)-10, identified by activation tagging, is a positive regulator of this process. When overexpressed (OE), CTG10 hastens aspects of seed germination. CTG10 is expressed predominantly in the hypocotyl, and the protein is localized to the nucleus. CTG10 interacts with PHYTOCHROME-INTERACTING FACTOR 1 (PIF1) and helps regulate its abundance in plantaCTG10-OE accelerates the loss of PIF1 in light, increasing germination efficiency, while PIF1-OE lines fail to complete germination in darkness, which is reversed by concurrent CTG10-OE Double-mutant (pif1 ctg10) lines demonstrated that PIF1 is epistatic to CTG10. Both CTG10 and PIF1 amounts decline during seed germination in the light but reaccumulate in the dark. PIF1 in turn down-regulates CTG10 transcription, suggesting a feedback loop of CTG10/PIF1 control. The genetic, physiological, and biochemical evidence, when taken together, leads us to propose that PIF1 and CTG10 coexist, and even accumulate, in the nucleus in darkness, but that, following illumination, CTG10 assists in reducing PIF1 amounts, thus promoting the completion of seed germination and subsequent seedling development.

A screen of activation tagged lines of Arabidopsis thaliana retrieved COLD TEMPERATURE GERMINATING10-D(tag) (CTG10-D(tag)) seeds, capable of radicle protrusion in advance of wild type (WT) at suboptimal- and optimal-temperatures. Genomic walking revealed T-DNA in the intragenic region that upregulates expression of the At4g19330 locus previously predicted to encode an F-BOX protein. A combination of surrogate splicing, primer scanning, RACE, and Illumina PolyAdenylation Tag (PAT) sequencing in petunia (Petunia X hybrida) and Arabidopsis were required to demonstrate that the region around At4g19330 was misannotated and is actually compromised of two separate genes. Even though homologous regions nearby and elsewhere on chromosome 4 are confounding elements in the molecular characterization of the locus, we could determine that the 5′ entity encodes a ribonucleoprotein of unknown function whereas the 3′ gene includes the promoter and full coding region of an F-BOX protein. Although both genes were upregulated, only independently-transformed lines over-expressing the F-Box exhibited enhanced completion of seed germination. We named it CTG10, and a single, poorly penetrant, mutant line of ctg10 manifested the expected reduced completion of seed germination. Whereas CTG10-OE lines are hyposensitive to the gibberellin biosynthetic inhibitor paclobutrazol, the ctg10 mutant line is hypersensitive; a phenotype which could be alleviated when transgenically rescued with CTG10. The F-Box moiety promoted association of CTG10 with ASK proteins in yeast two hybrid assays, indicating that it likely assembles into an SCF-type ubiquitin ligase to promote the ubiquitination of one or more substrates. In this capacity CTG10 might target a protein repressing seed germination for polyubiquitination and subsequent proteasomal degradation.

Members of the Bric-a-Brac/Tramtrack/Broad Complex (BTB) family direct the selective ubiquitylation of proteins following their assembly into Cullin3-based ubiquitin ligases. Here, we describe a subfamily of nucleus-localized BTB proteins encoded by the LIGHT-RESPONSE BTB1 (LRB1) and LRB2 loci in Arabidopsis (Arabidopsis thaliana) that strongly influences photomorphogenesis. Whereas single lrb1 and lrb2 mutants are relatively normal phenotypically, double mutants are markedly hypersensitive to red light, but not to far-red or blue light, and are compromised in multiple photomorphogenic processes, including seed germination, cotyledon opening and expansion, chlorophyll accumulation, shade avoidance, and flowering time. This red light hypersensitivity can be overcome by eliminating phytochrome B (phyB) and phyD, indicating that LRB1/2 act downstream of these two photoreceptor isoforms. Levels of phyB/D proteins but not their messenger RNAs are abnormally high in light-grown lrb1 lrb2 plants, implying that their light-dependent turnover is substantially dampened. Whereas other red light-hypersensitive mutants accumulate phyA protein similar to or higher than the wild type in light, the lrb1 lrb2 mutants accumulate less, suggesting that LRB1/2 also positively regulate phyA levels in a phyB/D-dependent manner. Together, these data show that the BTB ubiquitin ligases assembled with LRB1/2 function redundantly as negative regulators of photomorphogenesis, possibly by influencing the turnover of phyB/D.

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.

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.

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.

Selective modification of proteins by ubiquitination is directed by diverse families of ubiquitin-protein ligases (or E3s). A large collection of E3s use Cullins (CULs) as scaffolds to form multisubunit E3 complexes in which the CUL binds a target recognition subcomplex and the RBX1 docking protein, which delivers the activated ubiquitin moiety. Arabidopsis and rice contain a large collection of CUL isoforms, indicating that multiple CUL-based E3s exist in plants. Here we show that Arabidopsis CUL3a and CUL3b associate with RBX1 and members of the broad complex/tramtrack/bric-a-brac (BTB) protein family to form BTB E3s. Eighty genes encoding BTB domain-containing proteins were identified in the Arabidopsis genome, indicating that a diverse array of BTB E3s is possible. In addition to the BTB domain, the encoded proteins also contain various other interaction motifs that likely serve as target recognition elements. DNA microarray analyses show that BTB genes are expressed widely in the plant and that tissue-specific and isoform-specific patterns exist. Arabidopsis defective in both CUL3a and CUL3b are embryo-lethal, indicating that BTB E3s are essential for plant development.

Ubiquitination of various intracellular proteins by ubiquitin-protein ligases (or E3s) plays an essential role in eukaryotic cell regulation primarily through its ability to selectively target proteins for degradation by the 26S proteasome. Skp1, Cullin, F-box (SCF) complexes are one influential E3 class that use F-box proteins to deliver targets to a core ligase activity provided by the Skp1, Cullin, and Rbx1 subunits. Almost 700 F-box proteins can be found in Arabidopsis, indicating that SCF E3s likely play a pervasive role in plant physiology and development. Here, we describe the reverse genetic analysis of two F-box proteins, EBF1 and -2, that work coordinately in SCF complexes to repress ethylene action. Mutations in either gene cause hypersensitivity to exogenous ethylene and its precursor 1-aminocyclopropane-1-carboxylic acid. EBF1 and -2 interact directly with ethylene insensitive 3 (EIN3), a transcriptional regulator important for ethylene signaling. Levels of EIN3 are increased in mutants affecting either EBF1 or -2, suggesting that the corresponding SCF complexes work together in EIN3 breakdown. Surprisingly, double ebf1 ebf2 mutants display a substantial arrest of seedling growth and have elevated EIN3 levels, even in the absence of exogenous ethylene. Collectively, our results show that the SCF(EBF1/EBF2)-dependent ubiquitination and subsequent removal of EIN3 is critical not only for proper ethylene signaling but also for growth in plants.

Attachment of one or more ubiquitins (Ubs) to various intracellular proteins has a number of roles in plants including the selective removal of regulatory proteins by the 26S proteasome. The final step in this modification is performed by ubiquitin-protein ligases (E3s) that promote Ub transfer to appropriate targets. One important family of E3s is defined by the presence of a HECT domain, an active site first found at the C-terminus of the human E3 (E6-AP). Using a consensus HECT domain as the query, we identified a family of seven HECT-containing ubiquitin-protein ligases (UPL1-UPL7) in Arabidopsis thaliana that can be grouped into four subfamilies. The UPL3 and UPL4 subfamily encodes approximately 200-kDa proteins with four Armadillo repeats similar to those in the nuclear pore protein importin-alpha, suggesting that these E3s identify their targets through binding to nuclear localization sequences. Although T-DNA disruptions of the UPL3 locus do not affect overall growth and development of Arabidopsis, the mutants show aberrant trichome morphology. Instead of developing three branches, many upl3 trichomes contain five or more branches. The upl3 trichomes also often undergo an additional round of endoreplication resulting in enlarged nuclei with ploidy levels of up to 64C. upl3 plants are hypersensitive to gibberellic acid-3 (GA3), consistent with the role of gibberellins in trichome development. The phenotype of upl3 mutants is similar to that of kaktus, a previously described set of trichome mutants with supernumerary branches. Genetic analyses confirmed that upl3 mutants and kaktus-2 are allelic with kaktus-2 plants harboring a splice-site mutation within the UPL3-transcribed region. Collectively, the data indicate that the ubiquitination of one or more activator proteins by UPL3 is necessary to repress excess branching and endoreplication of Arabidopsis trichomes.

A global analysis of gene expression events during shoot development in Arabidopsis was conducted using oligonucleotide array analysis. Shoots can be induced in tissue culture by preincubating root explants on an auxin-rich callus induction medium (CIM) and by transferring explants to a cytokinin-rich shoot induction medium (SIM), during which time explants become committed to shoot formation and ultimately form shoots. Oligonucleotide array data obtained during shoot development from ∼8000 Arabidopsis genes were subjected to principal component analysis, which demonstrated that the major components of variation in gene expression during shoot development can be represented by groups of genes, each group of which is upregulated at only one developmental stage. Two percent to three percent of the ∼8000 Arabidopsis genes monitored in this study were upregulated by fourfold or more at any one stage during shoot development. When upregulated and downregulated genes were categorized by function, it was observed that numerous hormone response genes were upregulated during preincubation on CIM. Groups of genes involved in signaling and/or transcription were induced at or before the time of shoot commitment, and genes that encode components of the photosynthetic apparatus were upregulated later in development before shoot emergence. Primary hormone response genes, such as Aux/IAA genes, were upregulated during preincubation on auxin-rich CIM, and cytokinin-responsive response regulator genes were upregulated during incubation on cytokinin-rich SIM. The expression of ARABIDOPSIS RESPONSE REGULATOR5, a type-A response regulator gene, was upregulated at the time of shoot commitment, and its expression was localized to sites of presumptive shoot formation. Two “hybrid” His kinases involved in cytokinin responses, CRE1, which encodes a cytokinin receptor, and CKI1, a gene that is capable of conferring cytokinin-independent shoot development, were upregulated during incubation on SIM.