A recessive mutation in the RUB1-conjugating enzyme, RCE1, reveals a requirement for RUB modification for control of ethylene biosynthesis and proper induction of basic chitinase and PDF1.2 in Arabidopsis

By screening etiolated Arabidopsis seedlings for mutants with aberrant ethylene-related phenotypes, we identified a mutant that displays features of the ethylene-mediated triple response even in the absence of ethylene. Further characterization showed that the phenotype observed for the dark-grown seedlings of this mutant is reversible by prevention of ethylene perception and is dependent on a modest increase in ethylene production correlated with an increase in 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) activity in the hypocotyl. Molecular characterization of leaves of the mutant revealed severely impaired induction of basic chitinase (chiB) and plant defensin (PDF)1.2 following treatment with jasmonic acid and/or ethylene. Positional cloning of the mutation resulted in identification of a 49-bp deletion in RCE1 (related to ubiquitin 1 (RUB1)-conjugating enzyme), which has been demonstrated to be responsible for covalent attachment of RUB1 to the SCF (Skpl Cdc 53 F-box) ubiquitin ligase complex to modify its activity. Our analyses with rce1-2 demonstrate a previously unknown requirement for RUB1 modification for regulation of ethylene biosynthesis and proper induction of defense-related genes in Arabidopsis.     

Mutation of E1-CONJUGATING ENZYME-RELATED1 decreases RELATED TO UBIQUITIN conjugation and alters auxin response and development

The ubiquitin-like protein RELATED TO UBIQUITIN (RUB) is conjugated to CULLIN (CUL) proteins to modulate the activity of Skp1-Cullin-F-box (SCF) ubiquitylation complexes. RUB conjugation to specific target proteins is necessary for the development of many organisms, including Arabidopsis (Arabidopsis thaliana). Here, we report the isolation and characterization of e1-conjugating enzyme-related1-1 (ecr1-1), an Arabidopsis mutant compromised in RUB conjugation. The ecr1-1 mutation causes a missense change located two amino acid residues from the catalytic site cysteine, which normally functions to form a thioester bond with activated RUB. A higher ratio of unmodified CUL1 relative to CUL1-RUB is present in ecr1-1 compared to wild type, suggesting that the mutation reduces ECR1 function. The ecr1-1 mutant is resistant to the auxin-like compound indole-3-propionic acid, produces fewer lateral roots than wild type, displays reduced adult height, and stabilizes a reporter fusion protein that is degraded in response to auxin, suggesting reduced auxin signaling in the mutant. In addition, ecr1-1 hypocotyls fail to elongate normally when seedlings are grown in darkness, a phenotype shared with certain other RUB conjugation mutants that is not general to auxin-response mutants. The suite of ecr1-1 molecular and morphological phenotypes reflects roles for RUB conjugation in many aspects of plant growth and development. Certain ecr1-1 elongation defects are restored by treatment with the ethylene-response inhibitor silver nitrate, suggesting that the short ecr1-1 root and hypocotyl result from aberrant ethylene accumulation. Further, silver nitrate supplementation in combination with various auxins and auxin-like compounds reveals that members of this growth regulator family may differentially rely on ethylene signaling to inhibit root growth.     

Role of the Arabidopsis RING-H2 protein RBX1 in RUB modification and SCF function

The ubiquitin-related protein RUB/Nedd8 is conjugated to members of the cullin family of proteins in plants, animals, and fungi. In Arabidopsis, the RUB conjugation pathway consists of a heterodimeric E1 (AXR1-ECR1) and a RUB-E2 called RCE1. The cullin CUL1 is a subunit in SCF-type ubiquitin protein ligases (E3s), including the SCF(TIR1) complex, which is required for response to the plant hormone auxin. Our previous studies showed that conjugation of RUB to CUL1 is required for normal SCF(TIR1) function. The RING-H2 finger protein RBX1 is a subunit of SCF complexes in fungi and animals. The function of RBX1 is to bind the ubiquitin-conjugating enzyme E2 and bring it into close proximity with the E3 substrate. We have identified two Arabidopsis genes encoding RING-H2 proteins related to human RBX1. Studies of one of these proteins indicate that, as in animals and fungi, Arabidopsis RBX1 is an SCF subunit. Reduced RBX1 levels result in severe defects in growth and development. Overexpression of RBX1 increases RUB modification of CUL1. This effect is associated with reduced auxin response and severe growth defects similar to those observed in axr1 mutants. As in the axr1 mutants, RBX1 overexpression stabilizes the SCF(TIR1) substrate AXR2/IAA7. The RBX1 protein is a component of SCF complexes in Arabidopsis. In addition to its direct role in SCF E3 ligase activity, RBX1 promotes the RUB modification of CUL1 and probably functions as an E3 ligase in the RUB pathway. Hypermodification of CUL1 disrupts SCF(TIR1) function, suggesting that cycles of RUB conjugation and removal are important for SCF activity.     

Arabidopsis DCP2, DCP1, and VARICOSE form a decapping complex required for postembryonic development

mRNA turnover in eukaryotes involves the removal of m7GDP from the 5' end. This decapping reaction is mediated by a protein complex well characterized in yeast and human but not in plants. The function of the decapping complex in the development of multicellular organisms is also poorly understood. Here, we show that Arabidopsis thaliana DCP2 can generate from capped mRNAs, m7GDP, and 5'-phosphorylated mRNAs in vitro and that this decapping activity requires an active Nudix domain. DCP2 interacts in vitro and in vivo with DCP1 and VARICOSE (VCS), an Arabidopsis homolog of human Hedls/Ge-1. Moreover, the interacting proteins stimulate DCP2 activity, suggesting that the three proteins operate as a decapping complex. Consistent with their role in mRNA decay, DCP1, DCP2, and VCS colocalize in cytoplasmic foci, which are putative Arabidopsis processing bodies. Compared with the wild type, null mutants of DCP1, DCP2, and VCS accumulate capped mRNAs with a reduced degradation rate. These mutants also share a similar lethal phenotype at the seedling cotyledon stage, with disorganized veins, swollen root hairs, and altered epidermal cell morphology. We conclude that mRNA turnover mediated by the decapping complex is required for postembryonic development in Arabidopsis.     

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.     

Light Regulates the RUBylation Levels of Individual Cullin Proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In plants, the small protein related to ubiquitin (RUB) modifies cullin (CUL) proteins in ubiquitin E3 ligases to allow for efficient transfer of ubiquitin to substrate proteins for degradation by the 26S proteasome. At the molecular level, the conjugation of RUB to individual CUL proteins is transient in nature, which aids in the stability of the cullins and adaptor proteins. Many changes in cellular processes occur within the plant upon exposure to light, including well-documented changes in the stability of individual proteins. However, overall activity of E3 ligases between dark- and light-grown seedlings has not been assessed in plants. In order to understand more about the activity of the protein degradation pathway, overall levels of RUB-modified CULs were measured in Arabidopsis thaliana seedlings growing in different light conditions. We found that light influenced the global levels of RUBylation on CULs, but not uniformly. Blue light had little effect on both Cul1 and Cul3 RUBylation levels. However, red light directed the increase in Cul3 RUBylation levels, but not Cul1. This red-light regulation of Cul3 was at least partially dependent on the activation of the phytochrome B signaling pathway. The results indicate that the RUBylation levels on individual CULs change in response to different light conditions, which enable plants to fine-tune their growth and development to the various light environments.     

The RUB/Nedd8 conjugation pathway is required for early development in Arabidopsis

The related-to-ubiquitin (RUB) protein is post-translationally conjugated to the cullin subunit of the SCF (SKP1, Cullin, F-box) class of ubiquitin protein ligases. Although the precise biochemical function of RUB modification is unclear, studies indicate that the modification is important for SCF function. In Arabidopsis, RUB modification of CUL1 is required for normal function of SCF(TIR1), an E3 required for response to the plant hormone auxin. In this report we show that an Arabidopsis protein called RCE1 functions as a RUB-conjugating enzyme in vivo. A mutation in the RCE1 gene results in a phenotype like that of the axr1 mutant. Most strikingly, plants deficient in both RCE1 and AXR1 have an embryonic phenotype similar to mp and bdl mutants, previously shown to be deficient in auxin signaling. Based on these results, we suggest that the RUB-conjugation pathway is required for auxin-dependent pattern formation in the developing embryo. In addition, we show that RCE1 interacts directly with the RING protein RBX1 and is present in a stable complex with SCF. We propose that RBX1 functions as an E3 for RUB modification of CUL1.