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Indole-3-carbinol (I3C), a hydrolysis product of indole-3-methylglucosinolate, is toxic to herbivorous insects and pathogens. In mammalians, I3C is extensively studied for its properties in cancer prevention and treatment. Produced in Brassicaceae, I3C reversibly inhibits root elongation in a concentration-dependent manner. This inhibition is partially explained by the antagonistic action of I3C on auxin signaling through TIR1. To further elucidate the mode-of-action of I3C in plants, we have identified and characterized a novel Arabidopsis mutant tolerant to I3C, ICT1. This mutant was identified following screening of the Full-length cDNA Over-eXpression library (FOX) seed collection for root growth in the presence of exogenous I3C. ICT1 carries the AT2G19750 gene which encodes a S30 ribosomal protein. Overexpression of S30 gene and not the knockout of the underlying gene causes tolerance to I3C. The tolerance is specific to I3C, since ICT1 did not exhibit pronounced tolerance to other indol or benzoxanoids molecules tested. ICT1 maintains I3C-induced antagonism of auxin signaling, indicating that the tolerance is due to an auxin-independent mechanism. Transcript profiling experiments revealed that ICT1 is transcriptionally primed to respond to I3C treatment.

The COP9 signalosome (CSN) is a multisubunit regulator highly conserved in evolution. We show here that CSN subunit 8 (CSN8) is essential for Drosophila development. CSN8 is maternally contributed and present throughout development. Null mutants generated in this study are larval lethal, showing phenotypes associated with mutations in either CSN4 (molting defects) or CSN5 (melanotic tumors). Analysis of mitotic and germ-line csn8(null) clones revealed the requirement of CSN8 for multiple developmental processes. The germ-line clones arrested at mid-oogenesis, while the mitotic clones led to deformed adult eyes or wings. CSN8 is present exclusively as part of the CSN holo-complex, and lack of CSN8 in the mutants leads to CSN instability. Consistent with this, Cullin deneddylation is impaired in the csn8(null) mutants.

The roles of individual eukaryotic translation initiation factor 3 (eIF3) subunits are largely unclear, although some are essential, while others are thought to have regulatory roles. The 'e' subunit, also known as Int-6/Int6, is a candidate for a regulatory subunit as it is not essential for translation initiation in yeasts. eIF3e associates with the COP9 signalosome, and localizes to the nucleus in certain tissues. To further elucidate the roles of eIF3e, we have taken a genetic approach using Arabidopsis as a model system. Overexpression of eIF3e results in defects similar to mutations in the COP9 signalosome. eIF3e protein, but not transcript, over accumulates in csn mutants, and eIF3e is degraded in a proteasome-dependent fashion. In vitro and in vivo assays suggest that excess eIF3e inhibits translation. We conclude that the COP9 signalosome maintains a precise regulation of eIF3e levels, which is necessary for normal development in Arabidopsis.

The COP9 signalosome is a multifunctional regulator essential for Drosophila development. A loss-of-function mutant in Drosophila COP9 signalosome subunit 5 (CSN5) develops melanotic bodies, a phenotype common to mutants in immune signaling. csn5(null) larvae accumulated high levels of Cactus that co-localizes with Dorsal to the nucleus. However, Dorsal-dependent transcriptional activity remained repressed in the absence of an inducing signal, despite its nuclear localization. Dorsal activity in mutant larvae and NFkappaB activity in CSN5 down-regulated mammalian cells can be induced following activation of the Toll/IL-1 pathway. csn5(null) larvae contained more hemocytes than wild-type (wt) larvae. A large portion of these cells have differentiated to lamellocytes (LM), a hemocyte cell type rarely seen in normal larvae. The results presented here indicate that CSN5 is a negative regulator of Dorsal subcellular localization, and of hemocyte proliferation and differentiation. These results further indicate that nuclear localization of Dorsal can be uncoupled from its activation. Surprisingly, CSN5 is not necessary for immune-induced degradation of Cactus.

The COP9 signalosome (CSN) is a multisubunit protein complex that intersects the ubiquitin (Ub)-proteasome pathway at several junctions. The CSN associates with several protein kinases that target key regulatory proteins that themselves are targets for Ub-dependent degradation, and several subunits themselves are phosphoproteins. We previously reported that Arabidopsis CSN7 is a phosphoprotein. To identify the kinases responsible for CSN7 phosphorylation, we biochemically purified CSN7-kinsae activities from cauliflower through a four-step purification procedure that resulted in a ca. 300-fold enrichment. One of these activities is Ca 2+ dependent, while the second is not. Peptide fingerprint analysis of the purified proteins identified three putative CSN7 kinases.

The Arabidopsis COP9 complex is a multi-subunit repressor of photomorphogenesis which is conserved among multicellular organisms. Approximately 12 proteins copurify with the COP9 complex. Seven of these proteins are orthologues of subunits of the recently published mammalian COP9 complex. Four of the proteins show amino acid similarity to various subunits of the COP9 complex, eIF3 complex and 19S cap of the proteasome. We have studied one of these proteins in order to determine if it is a component of the COP9 complex. Arabidopsis p105 is highly similar to the p110 subunit of the human eIF3. The p105 gene is induced during photomorphogenesis, and RNA and protein analysis reveal different tissue accumulation patterns. p105 is found in a large protein complex. p105 interacts in yeast with both COP9 and FUS6, two known components of the COP9 complex. Our results indicate that p105 is not a component of the COP9 core complex, though it may interact with components of the complex.

Light signals perceived by photoreceptors are transduced into the nucleus to regulate gene expression and development. The COP9 protein complex localized in the nucleus has a central role in modulating light-regulated development, and may have a role in regulating development in general in all organisms. This review considers recent advances in the study of the COP9 complex and its sub-units in plant and animal systems, and discusses the possible functional implications.

Carotenoids with cyclic end groups are essential components of the photosynthetic membrane in all known oxygenic photosynthetic organisms. These yellow pigments serve the vital role of protecting against potentially lethal photo-oxidative damage. Many of the enzymes and genes of the carotenoid biosynthetic pathway in cyanobacteria, algae and plants remain to be isolated or identified. We have cloned a cyanobacterial gene encoding lycopene cyclase, an enzyme that converts the acyclic carotenoid lycopene to the bicyclic molecule beta-carotene. The gene was identified through the use of an experimental herbicide, 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA), that prevents the cyclization of lycopene in plants and cyanobacteria. Chemically-induced mutants of the cyanobacterium Synechococcus sp. PCC7942 were selected for resistance to MPTA, and a mutation responsible for this resistance was mapped to a genomic DNA region of 200 bp by genetic complementation of the resistance in wild-type cells. A 1.5 kb genomic DNA fragment containing this MPTA-resistance mutation was expressed in a lycopene-accumulating strain of Escherichia coli. The conversion of lycopene to beta-carotene in these cells demonstrated that this fragment encodes the enzyme lycopene cyclase. The results indicate that a single gene product, designated lcy, catalyzes both of the cyclization reactions that are required to produce beta-carotene from lycopene, and prove that this enzyme is a target site of the herbicide MPTA. The cloned cyanobacterial lcy gene hybridized well with genomic DNA from eukaryotic algae, thus it will enable the identification and cloning of homologous genes for lycopene cyclase in algae and plants.

Protein kinase transduction pathways are thought to be involved in light signaling in plants, but other than the photoreceptors, no protein kinase activity has been shown to be light-regulated in vivo. Using an in-gel protein kinase assay technique with histone H III SS as an exogenous substrate, we identified a light-regulated protein kinase activity with an apparent molecular weight ca 50 kDa. The kinase activity increased transiently after irradiation of dark-grown seedlings with continuous far red light (FR) and blue light (B) and decreased after irradiation with red light (R). The maximal activation was achieved after 30 min to 1 h with FR or B. After irradiation times longer than 2 h, the kinase activity decreased to below the sensitivity level of the assay. In Arabidopsis mutants lacking either the photoreceptors phytochrome A, phytochrome B or the blue-light receptor cryptochrome 1, kinase activity was undetectable, whereas in the photomorphogenic mutants cop1 and det1 the kinase activity was also observed in the absence of light signals, though still stimulated by B and FR. Interestingly, the R inhibition of the kinase activity was lost in the mutant hy5. Pretreatment with cycloheximide blocked the kinase activity.