The ubiquitin-specific protease UBP14 is essential for early embryo development in Arabidopsis thaliana.

The ubiquitin/26S proteasome pathway is a major route for selectively degrading cytoplasmic and nuclear proteins in eukaryotes. In this pathway, chains of ubiquitins become attached to short-lived proteins, signalling recognition and breakdown of the modified protein by the 26S proteasome. During or following target degradation, the attached multi-ubiquitin chains are released and subsequently disassembled by ubiquitin-specific proteases (UBPs) to regenerate free ubiquitin monomers for re-use. Here, we describe Arabidopsis thaliana UBP14 that may participate in this recycling process. Its amino acid sequence is most similar to yeast UBP14 and its orthologues, human IsoT1-3 and Dictyostelium UbpA, and it can functionally replace yeast UBP14 in a ubp14Delta mutant. Like its orthologues, AtUBP14 can disassemble multi-ubiquitin chains linked internally via epsilon-amino isopeptide bonds using Lys48 and can process some, but not all, translational fusions of ubiquitin linked via alpha-amino peptide bonds. However, unlike its yeast and Dictyostelium orthologues, AtUBP14 is essential in Arabidopsis. T-DNA insertion mutations in the single gene that encodes AtUBP14 cause an embryonic lethal phenotype, with the homozygous embryos arresting at the globular stage. The arrested seeds have substantially increased levels of multi-ubiquitin chains, indicative of a defect in ubiquitin recycling. Taken together, the data demonstrate an essential role for the ubiquitin/26S proteasome pathway in general and for AtUBP14 in particular during early plant development.

The cellular level of PR500, a protein complex related to the 19S regulatory particle of the proteasome, is regulated in response to stresses in plants.

In Arabidopsis seedlings and cauliflower florets, Rpn6 (a proteasome non-ATPase regulatory subunit) was found in two distinct protein complexes of approximately 800 and 500 kDa, respectively. The large complex likely represents the proteasome 19S regulator particle (RP) because it displays the expected subunit composition and all characteristics. The small complex, designated PR500, shares at least three subunits with the "lid" subcomplex of 19S RP and is loosely associated with an hsp70 protein. In Arabidopsis COP9 signalosome mutants, PR500 was specifically absent or reduced to an extent that correlates with the severity of the mutations. Furthermore, PR500 was also diminished in response to potential protein-misfolding stresses caused by the heat shock and canavanine treatment. Immunofluorescence studies suggest that PR500 has a distinct localization pattern and is enriched in specific nuclear foci. We propose that PR500 may be evolved in higher plants to cope with the frequently encountered environmental stresses.

The Arabidopsis thaliana HY1 locus, required for phytochrome-chromophore biosynthesis, encodes a protein related to heme oxygenases.

The hy1 mutants of Arabidopsis thaliana fail to make the phytochrome-chromophore phytochromobilin and therefore are deficient in a wide range of phytochrome-mediated responses. Because this defect can be rescued by feeding seedlings biliverdin IXalpha, it is likely that the mutations affect an enzyme that converts heme to this phytochromobilin intermediate. By a combination of positional cloning and candidate-gene isolation, we have identified the HY1 gene and found it to be related to cyanobacterial, algal, and animal heme oxygenases. Three independent alleles of hy1 contain DNA lesions within the HY1 coding region, and a genomic sequence spanning the HY1 locus complements the hy1-1 mutation. HY1 is a member of a gene family and is expressed in a variety of A. thaliana tissues. Based on its homology, we propose that HY1 encodes a higher-plant heme oxygenase, designated AtHO1, responsible for catalyzing the reaction that opens the tetrapyrrole ring of heme to generate biliverdin IXalpha.

Polyamines and paraquat toxicity in Arabidopsis thaliana.

The relationship between paraquat toxicity and polyamine levels was analyzed in Arabidopsis wild-type and gi-3 plants. Paraquat treatment led to an increase in putrescine, but not of spermidine and spermine content. Additionally, polyamine feeding offered high levels of protection against paraquat toxicity with spermidine being the most effective protectant.

Oxidative stress tolerance and longevity in Arabidopsis: the late-flowering mutant gigantea is tolerant to paraquat.

Recent genetic analyses of longevity in animals have revealed that long-lived strains are more tolerant to environmental stresses. To investigate whether extended longevity in Arabidopsis also correlates with an increase in stress tolerance, the response was tested of 11 late-flowering mutants to the superoxide radical-generating herbicide paraquat. A tight correlation between flowering time and paraquat tolerance was found when plants were exposed to low doses of herbicide. Furthermore, the mutant gigantea (gi-3) with the longest delay in flowering time had a high tolerance level to paraquat-induced oxidative stress. All the tested gi alleles had an increased tolerance to paraquat toxicity compared to wild-type, although the actual levels of tolerance differed. In addition, the gi-3 mutant was more tolerant to hydrogen peroxide. These results suggest that the link between longevity and oxidative stress resistance in plants is similar to that found in animals, implying that this phenomenon may be general for all aerobic organisms.

The trihelix DNA-binding motif in higher plants is not restricted to the transcription factors GT-1 and GT-2.

GT-2 is a plant transcriptional activator that contains two separate, but similar, trihelix DNA-binding domains. GT-1 is similar to GT-2, although it contains only one of such domains. cDNAs that encode GT-2 were isolated from rice (OS-GT2) and Arabidopsis (AT-GT2). Evidence is presented for the existence of an Arabidopsis gene family that is structurally related to AT-GT2. Two members of this GT2-like family, AT-GTL1 and AT-GTL2, have been isolated and characterized. Their sequences suggest that they evolved by a recent gene duplication event. Both AT-GT2 and AT-GTL genes contain an intron in the amino-terminal trihelix motif, indicating that this DNA-binding domain resulted from exon shuffling. RNA gel blot analysis using AT-GTL1 as a probe revealed four transcripts in the aerial part of the plant. All mRNA levels were significantly higher in siliques, suggesting that this gene family may function in fruit and/or seed development. To date, DNA-binding proteins characterized by the trihelix motif have been described only in plants, and may therefore be involved in plant-specific processes. Our results show that in Arabidopsis thaliana, the trihelix motif is not restricted to the GT-1 and GT-2 DNA-binding proteins.

Differential expression of CuZn- and Fe-superoxide dismutase genes of tobacco during development, oxidative stress, and hormonal treatments.

Chloroplasts of Nicotiana tabacum have two superoxide dismutases: a Fe- and a CuZn-containing enzyme, encoded by the nuclear genes sodB and sodCp, respectively. As a first step in studying the physiological function of these two enzymes, we compared the expression of sodB and sodCp in different plant organs, in response to hormonal treatments, and upon treatment with paraquat and Norflurazon. The sodCp transcript and active enzyme were detected only in young leaves of mature plants. The sodB transcript was more abundant in young compared to old leaves, but the enzymatic activity was higher in mature and senescent leaves. sodCp and sodB exhibited a different expression pattern upon treatment with abscisic acid, indole-3-acetic acid, kinetin, gibberellin, and 1-aminocyclopropane-1-carboxylate. Paraquat treatment caused a decrease in abundance of both transcripts, although the dose dependency of this decrease differed. Norflurazon-induced photooxidation resulted in a 10-fold increase of sodCp mRNA whereas the sodB transcript level was 25% higher than the control. These differences in expression might explain why both plastid-located superoxide dismutase enzymes are needed, particularly under stress conditions.

Ethylene can stimulate Arabidopsis hypocotyl elongation in the light.

Ethylene inhibits hypocotyl elongation in etiolated Arabidopsis seedlings. However, when Arabidopsis was grown in the light in the presence of ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), a marked induction of hypocotyl elongation occurred. This resulted from an increase in cell expansion rather than cell division. The effects of ethylene and ACC were antagonized by the ethylene action inhibitor Ag+. The elongation response was absent or weakened in a set of ethylene-insensitive mutants (etr1-3, ein2-1, ein3-1, ein4, ain1-10, ein7). With the exception of ein4, the degree of inhibition of hypocotyl elongation was correlated with the strength of the ethylene-insensitive phenotype based on the triple response assay. In addition, the constitutive ethylene response mutant ctr1-1, grown in the light, had a longer hypocotyl than the wild type. Exogenous auxin also induced hypocotyl elongation in light-grown Arabidopsis. Again, the response was abolished by treatment with Ag+, suggesting that ethylene might be a mediator. The results showed that, depending on light conditions, ethylene can induce opposite effects on cell expansion in Arabidopsis hypocotyls.