[Show abstract][Hide abstract] ABSTRACT: Ubiquitin C-terminal hydrolases (UCHs) are a subset of de-ubiquitinating proteases that release covalently linked ubiquitin (Ub), and as such play essential roles in recycling Ub and reversing the action of Ub conjugation. We show here that two related Arabidopsis UCHs, UCH1, and UCH2, are important for shoot development. The UCH1 and 2 genes are ubiquitously expressed, with the corresponding proteins present in both the cytoplasm and nucleus. Unlike their animal and fungal counterparts, we found no evidence that the Arabidopsis UCH1 and 2 proteins stably associate with the 26S proteasome. Altering the levels of UCH1 and 2 has substantial effects on Arabidopsis shoot development, especially with respect to inflorescence architecture, with over-expression and double mutants enhancing and suppressing the outgrowth of cauline branches, respectively. Neither UCH1-over-expressing nor uch1-1 uch2-1 plants have detectably altered sensitivity to cytokinins or auxins individually, but exhibit an altered sensitivity to the ratio of the two hormones. UCH1-over-expressing plants show dramatically enhanced phenotypes when combined with auxin-insensitive mutants axr1-3 and axr2-1, suggesting that one or more aspects of auxin signaling are affected by this enzyme pair. Previous studies revealed that the ubiquitination and degradation of the AUX/IAA family of repressors is a key step in auxin signaling. Here, we show that turnover of a reporter fused to a representative AUX/IAA protein AXR3 is faster in the uch1-1 uch2-1 double mutant but slower in the UCH1 over-expression backgrounds. Taken together, our results indicate that de-ubiquitination helps to modify plant shoot architecture, possibly via its ability to directly or indirectly protect upstream target proteins involved in auxin/cytokinin signaling from Ub-mediated degradation.
[Show abstract][Hide abstract] ABSTRACT: Ethylene signaling in Arabidopsis thaliana converges on the ETHYLENE-INSENSITIVE3 (EIN3)/EIN3-Like (EIL) transcription factors to induce various responses. EIN3 BINDING F-BOX1 (EBF1) and EBF2 were recently shown to function in ethylene perception by regulating EIN3/EIL turnover. In the absence of ethylene, EIN3 and possibly other EIL proteins are targeted for ubiquitination and subsequent degradation by Cullin 1-based E3 complexes containing EBF1 and 2. Ethylene appears to block this ubiquitination, allowing EIN3/EIL levels to rise and mediate ethylene signaling. Through analysis of mutant combinations affecting accumulation of EBF1, EBF2, EIN3, and EIL1, we show that EIN3 and EIL1 are the main targets of EBF1/2. Kinetic analyses of hypocotyl growth inhibition in response to ethylene and growth recovery after removal of the hormone revealed that EBF1 and 2 have temporally distinct but overlapping roles in modulating ethylene perception. Whereas EBF1 plays the main role in air and during the initial phase of signaling, EBF2 plays a more prominent role during the latter stages of the response and the resumption of growth following ethylene removal. Through their coordinated control of EIN3/EIL1 levels, EBF1 and EBF2 fine-tune ethylene responses by repressing signaling in the absence of the hormone, dampening signaling at high hormone concentrations, and promoting a more rapid recovery after ethylene levels dissipate.
[Show abstract][Hide abstract] ABSTRACT: The oxidative cleavage of heme by heme oxygenases (HOs) to form biliverdin IXalpha (BV) is the committed step in the biosynthesis of the phytochrome (phy) chromophore and thus essential for proper photomorphogenesis in plants. Arabidopsis (Arabidopsis thaliana) contains four possible HO genes (HY1, HO2-4). Genetic analysis of the HY1 locus showed previously that it is the major source of BV with hy1 mutant plants displaying long hypocotyls and decreased chlorophyll accumulation consistent with a substantial deficiency in photochemically active phys. More recent analysis of HO2 suggested that it also plays a role in phy assembly and photomorphogenesis but the ho2 mutant phenotype is more subtle than that of hy1 mutants. Here, we define the functions of HO3 and HO4 in Arabidopsis. Like HY1, the HO3 and HO4 proteins have the capacity to synthesize BV from heme. Through a phenotypic analysis of T-DNA insertion mutants affecting HO3 and HO4 in combination with mutants affecting HY1 or HO2, we demonstrate that both of the encoded proteins also have roles in photomorphogenesis, especially in the absence of HY1. Disruption of HO3 and HO4 in the hy1 background further desensitizes seedlings to red and far-red light and accelerates flowering time, with the triple mutant strongly resembling seedlings deficient in the synthesis of multiple phy apoproteins. The hy1/ho3/ho4 mutant can be rescued phenotypically and for the accumulation of holo-phy by feeding seedlings BV. Taken together, we conclude that multiple members of the Arabidopsis HO family are important for synthesizing the bilin chromophore used to assemble photochemically active phys.
[Show abstract][Hide abstract] ABSTRACT: The 26S proteasome is an essential protease complex responsible for removing most short-lived intracellular proteins, especially those modified with polyubiquitin chains. We show here that an Arabidopsis mutant expressing an altered RPN10 subunit exhibited a pleiotropic phenotype consistent with specific changes in 26S proteasome function. rpn10-1 plants displayed reduced seed germination, growth rate, stamen number, genetic transmission through the male gamete, and hormone-induced cell division, which can be explained partially by a constitutive downregulation of the key cell cycle gene CDKA;1. rpn10-1 also was more sensitive to abscisic acid (ABA), salt, and sucrose stress and to DNA-damaging agents and had decreased sensitivity to cytokinin and auxin. Most of the phenotypes can be explained by a hypersensitivity to ABA, which is reflected at the molecular level by the selective stabilization of the short-lived ABA-signaling protein ABI5. Collectively, these results indicate that RPN10 affects a number of regulatory processes in Arabidopsis likely by directing specific proteins to the 26S proteasome for degradation. A particularly important role may be in regulating the responses to signals promulgated by ABA.