The silencing of one parental set of rRNA genes in a genetic hybrid is an epigenetic phenomenon known as nucleolar dominance. We showed previously that silencing is restricted to the nucleolus organizer regions (NORs), the loci where rRNA genes are tandemly arrayed, and does not spread to or from neighboring protein-coding genes. One hypothesis is that nucleolar dominance is the net result of hundreds of silencing events acting one rRNA gene at a time. A prediction of this hypothesis is that rRNA gene silencing should occur independent of chromosomal location. An alternative hypothesis is that the regulatory unit in nucleolar dominance is the NOR, rather than each individual rRNA gene, in which case NOR localization may be essential for rRNA gene silencing. To test these alternative hypotheses, we examined the fates of rRNA transgenes integrated at ectopic locations. The transgenes were accurately transcribed in all independent transgenic Arabidopsis thaliana lines tested, indicating that NOR localization is not required for rRNA gene expression. Upon crossing the transgenic A. thaliana lines as ovule parents with A. lyrata to form F1 hybrids, a new system for the study of nucleolar dominance, the endogenous rRNA genes located within the A. thaliana NORs are silenced. However, rRNA transgenes escaped silencing in multiple independent hybrids. Collectively, our data suggest that rRNA gene activation can occur in a gene-autonomous fashion, independent of chromosomal location, whereas rRNA gene silencing in nucleolar dominance is locus-dependent.

Deubiquitinating enzymes are essential to the ubiquitin (Ub)/26S proteasome system where they release Ub monomers from the primary translation products of poly-Ub and Ub extension genes, recycle Ubs from polyubiquitinated proteins, and reverse the effects of ubiquitination by releasing bound Ubs from individual targets. The Ub-specific proteases (UBPs) are one large family of deubiquitinating enzymes that bear signature cysteine and histidine motifs. Here, we genetically characterize a UBP subfamily in Arabidopsis (Arabidopsis thaliana) encoded by paralogous UBP3 and UBP4 genes. Whereas homozygous ubp3 and ubp4 single mutants do not display obvious phenotypic abnormalities, double-homozygous mutant individuals could not be created due to a defect in pollen development and/or transmission. This pollen defect was rescued with a transgene encoding wild-type UBP3 or UBP4, but not with a transgene encoding an active-site mutant of UBP3, indicating that deubiquitination activity of UBP3/UBP4 is required. Nuclear DNA staining revealed that ubp3 ubp4 pollen often fail to undergo mitosis II, which generates the two sperm cells needed for double fertilization. Substantial changes in vacuolar morphology were also evident in mutant grains at the time of pollen dehiscence, suggesting defects in vacuole and endomembrane organization. Even though some ubp3 ubp4 pollen could germinate in vitro, they failed to fertilize wild-type ovules even in the absence of competing wild-type pollen. These studies provide additional evidence that the Ub/26S proteasome system is important for male gametogenesis in plants and suggest that deubiquitination of one or more targets by UBP3/UBP4 is critical for the development of functional pollen.

Autophagy is an important mechanism for nonselective intracellular breakdown whereby cytosol and organelles are encapsulated in vesicles, which are then engulfed and digested by lytic vacuoles/lysosomes. In yeast, this encapsulation employs a set of autophagy (ATG) proteins that direct the conjugation of two ubiquitin-like protein tags, ATG8 and ATG12, to phosphatidylethanolamine and the ATG5 protein, respectively. Using an Arabidopsis (Arabidopsis thaliana) atg7 mutant unable to ligate either tag, we previously showed that the ATG8/12 conjugation system is important for survival under nitrogen-limiting growth conditions. By reverse-genetic analyses of the single Arabidopsis gene encoding ATG5, we show here that the subpathway that forms the ATG12-ATG5 conjugate also has an essential role in plant nutrient recycling. Similar to plants missing ATG7, those missing ATG5 display early senescence and are hypersensitive to either nitrogen or carbon starvation, which is accompanied by a more rapid loss of organellar and cytoplasmic proteins. Multiple ATG8 isoforms could be detected immunologically in seedling extracts. Their abundance was substantially elevated in both the atg5 and atg7 mutants, caused in part by an increase in abundance of several ATG8 mRNAs. Using a green fluorescent protein-ATG8a fusion in combination with concanamycin A, we also detected the accumulation of autophagic bodies inside the vacuole. This accumulation was substantially enhanced by starvation but blocked in the atg7 background. The use of this fusion in conjunction with atg mutants now provides an important marker to track autophagic vesicles in planta.

Phospholipase A2s (PLA2s) are enzymes that liberate lysophospholipids and free fatty acids (FFAs) from membrane phospholipids in response to hormones and other external stimuli. This report describes the cloning and functional characterization of a PLA2 cDNA from Arabidopsis thaliana, AtsPLA2-alpha, which represents one of four secretory PLA2 (sPLA2) genes in Arabidopsis. The encoded protein is 148-amino acid polypeptide and is predicted to contain a 20-amino acid signal peptide at its amino terminus. The predicted mature form (Mr=14,169) of AtsPLA2-alpha exhibited approximately 5 times the specific activity of its pre-processed form. Different from animal sPLA2s, AtsPLA2-alpha showed a significant preference for the acyl group linoleic acid over palmitic acid in phospholipid hydrolysis. Like some animal sPLA2s, however, it has a slight preference for phosphatidylethanolamine over phosphatidylcholine as the substrate. The specific activity of AtsPLA2-alpha continuously increased as the Ca2+ concentration was increased to 10 mM, and the optimal pH range was very broad and biphasic between 6 and 11. AtsPLA2-alpha transcript was detected at low levels in roots, stems, leaves, and flowers but not in siliques.

Multiple secretory phospholipase A2 (sPLA2) genes have been identified in plants and encode isoforms with distinct regulatory and catalytic properties. Elucidation of this genetic and biochemical heterogeneity has provided important clues to the regulation and function of the individual enzymes. An increasing body of evidence shows that their lipid products, lysophospholipids and free fatty acids, mediate a variety of cellular responses, including plant growth, development, and responses to stress and defense. This review discusses the newly-acquired information on plant sPLA2s including the molecular and biochemical characteristics, and signaling functions of each isoform.

The gene coding for APG8a (At4g21980), a protein from Arabidopsis thaliana, is involved in the autophagy process. The protein is an interesting candidate for structure determination by NMR spectroscopy. Toward this end, APG8a fused to an N-terminal His-tag has been expressed in Escherichia coli under a T7 expression system, refolded in vitro, and kept soluble by slight destabilization. The expressed protein appeared in both the soluble and the insoluble fractions. The whole-cell lysate was denatured by the addition of guanidinium chloride. The protein was immobilized on nickel–agarose resin and refolded by stepwise decrement of the denaturant. The elution buffer was 20mM sodium phosphate, pH 7.0, with 1% glycerol, 0.5M urea, 300mM NaCl, and 1M imidazole. After the removal of imidazole by ultrafiltration, the His-tag was cleaved with biotinylated thrombin. The protein product was kept in 20mM sodium phosphate, pH 7.0, with 1% glycerol, 0.5M urea, and 300mM NaCl. The protein was found to aggregate extensively over time if any one of the three ingredients (sodium chloride, urea, or glycerol) was omitted. The yield of the protein was around 20mg/L Luria–Bertani culture medium. The 1H–15N NMR correlation spectrum of 15N-labeled APG8a showed the characteristic signature of a folded protein; thus, the solutes appear to have no deleterious effect on the sample. These solution conditions kept the protein soluble and unaggregated for at least 2 days (enough time for NMR data collection). This approach of balanced stabilization–destabilization may offer a general approach for structural investigations of proteins that tend to aggregate.

The vacuole/lysosome serves an important recycling function during starvation and senescence in eukaryotes via a process called autophagy. Here bulk cytosolic constituents and organelles become sequestered in specialized autophagic vesicles, which then deliver their cargo to the vacuole for degradation. In yeasts, genetic screens have identified two novel post-translational modification pathways remarkably similar to ubiquitination that are required for autophagy. From searches of the Arabidopsis genome, we have identified gene families encoding proteins related to both the APG8 and -12 polypeptide tags and orthologs for all components required for their attachment. A single APG7 gene encodes the ATP-dependent activating enzyme that initiates both conjugation pathways. Phenotypic analysis of an APG7 disruption indicates that it is not essential for normal growth and development in Arabidopsis. However, the apg7-1 mutant is hypersensitive to nutrient limiting conditions and displays premature leaf senescence. mRNAs for both APG7 and APG8 preferentially accumulate as leaves senesce, suggesting that both conjugation pathways are up-regulated during the senescence syndrome. These findings show that the APG8/12 conjugation pathways have been conserved in plants and may have important roles in autophagic recycling, especially during situations that require substantial nitrogen and carbon mobilization.

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.

Ubiquitin-specific proteases (UBPs) are a family of unique hydrolases that specifically remove polypeptides covalently linked via peptide or isopeptide bonds to the C-terminal glycine of ubiquitin. UBPs help regulate the ubiquitin/26S proteolytic pathway by generating free ubiquitin monomers from their initial translational products, recycling ubiquitins during the breakdown of ubiquitin-protein conjugates, and/or by removing ubiquitin from specific targets and thus presumably preventing target degradation. Here, we describe a family of 27 UBP genes from Arabidopsis that contain both the conserved cysteine (Cys) and histidine boxes essential for catalysis. They can be clustered into 14 subfamilies based on sequence similarity, genomic organization, and alignments with their closest relatives from other organisms, with seven subfamilies having two or more members. Recombinant AtUBP2 functions as a bona fide UBP: It can release polypeptides attached to ubiquitins via either alpha- or epsilon-amino linkages by an activity that requires the predicted active-site Cys within the Cys box. From the analysis of T-DNA insertion mutants, we demonstrate that the AtUBP1 and 2 subfamily helps confer resistance to the arginine analog canavanine. This phenotype suggests that the AtUBP1 and 2 enzymes are needed for abnormal protein turnover in Arabidopsis.

As initial steps to define how the 26S proteasome degrades ubiquitinated proteins in plants, we have characterized many of the subunits that comprise the proteolytic complex from Arabidopsis thaliana. A set of 23 Arabidopsis genes encoding the full complement of core particle (CP) subunits and a collection encoding 12 out of 18 known eukaryotic regulatory particle (RP) subunits, including six AAA-ATPase subunits, were identified. Several of these 26S proteasome genes could complement yeast strains missing the corresponding orthologs. Using this ability of plant subunits to functionally replace yeast counterparts, a parallel structure/function analysis was performed with the RP subunit RPN10/MCB1, a putative receptor for ubiquitin conjugates. RPN10 is not essential for yeast viability but is required for amino acid analog tolerance and degradation of proteins via the ubiquitin-fusion degradation pathway, a subpathway within the ubiquitin system. Surprisingly, we found that the C-terminal motif required for conjugate recognition by RPN10 is not essential for in vivo functions. Instead, a domain near the N-terminus is required. We have begun to exploit the moss Physcomitrella patens as a model to characterize the plant 26S proteasome using reverse genetics. By homologous recombination, we have successfully disrupted the RPN10 gene. Unlike yeast rpn10Δ strains which grow normally, Physcomitrella rpn10Δ strains are developmentally arrested, being unable to initiate gametophorogenesis. Further analysis of these mutants revealed that RPN10 is likely required for a developmental program triggered by plant hormones.

The 26S proteasome is a multi-subunit ATP-dependent protease responsible for degrading most short-lived intracellular proteins targeted for breakdown by ubiquitin conjugation. The complex is composed of two relatively stable subparticles, the 20S proteasome, a hollow cylindrical structure which contains the proteolytic active sites in its lumen, and the 19S regulatory particle (RP) which binds to either end of the cylinder and provides the ATP-dependence and the specificity for ubiquitinated proteins. Among the approximately 18 subunits of the RP from yeast and animals are a set of six proteins, designated RPT1-6 for regulatory particle triple-A ATPase, that form a distinct family within the AAA superfamily. Presumably, these subunits use ATP hydrolysis to help assemble the 26S holocomplex, recognize and unfold appropriate substrates, and/or translocate the substrates to the 20S complex for degradation. Here, we describe the RPT gene family from Arabidopsis thaliana. From a collection of cDNAs and genomic sequences, a family of genes encoding all six of the RPT subunits was identified with significant amino acid sequence similarity to their yeast and animal counterparts. Five of the six RPT subunits are encoded by two genes; the exception being RPT3 which is encoded by a single gene. mRNA for each of the six proteins is present in all tissue types examined. Five of the subunits (RPT1 and 3-6) complemented yeast mutants missing their respective orthologs, indicating that the yeast and Arabidopsis proteins are functionally equivalent. Taken together, these results demonstrate that the RP, like the 20S proteasome, is functionally and structurally conserved among eukaryotes and indicate that the plant RPT subunits, like their yeast counterparts, have non-redundant functions.

The 20S proteasome is the proteolytic complex in eukaryotes responsible for degrading short-lived and abnormal intracellular proteins, especially those targeted by ubiquitin conjugation. The 700-kD complex exists as a hollow cylinder comprising four stacked rings with the catalytic sites located in the lumen. The two outer rings and the two inner rings are composed of seven different alpha and beta polypeptides, respectively, giving an alpha7/beta7/beta7/alpha7 symmetric organization. Here we describe the molecular organization of the 20S proteasome from the plant Arabidopsis thaliana. From an analysis of a collection of cDNA and genomic clones, we identified a superfamily of 23 genes encoding all 14 of the Arabidopsis proteasome subunits, designated PAA-PAG and PBA-PBG for Proteasome Alpha and Beta subunits A-G, respectively. Four of the subunits likely are encoded by single genes, and the remaining subunits are encoded by families of at least 2 genes. Expression of the alpha and beta subunit genes appears to be coordinately regulated. Three of the nine Arabidopsis proteasome subunit genes tested, PAC1 (alpha3), PAE1 (alpha5) and PBC2 (beta3), could functionally replace their yeast orthologs, providing the first evidence for cross-species complementation of 20S subunit genes. Taken together, these results demonstrate that the 20S proteasome is structurally and functionally conserved among eukaryotes and suggest that the subunit arrangement of the Arabidopsis 20S proteasome is similar if not identical to that recently determined for the yeast complex.

Rapid evolution of ribosomal RNA (rRNA) gene promoters often prevents their recognition in a foreign species. Unlike animal systems, we show that foreign plant rRNA gene promoters are recognized in an alien species, but tend to program transcription by a different polymerase. In plants, RNA polymerase I transcripts initiate at a TATATA element (+1 is underlined) important for promoter strength and start-site selection. However, transcripts initiate from +32 following transfection of a tomato promoter into Arabidopsis. The rRNA gene promoter of a more closely related species, Brassica oleracea, programs both +1 and +29 transcription. A point mutation at +2 improving the identity between the Brassica and Arabidopsis promoters increases +1 transcription, indicating a role for the initiator element in species-specificity. Brassica +29 transcripts can be translated to express a luciferase reporter gene, implicating RNA polymerase II. TATA mutations that disrupt TATA-binding protein (TBP) interactions inhibit +29 transcription and luciferase expression. Co-expressed TBP proteins bearing compensatory mutations restore +29 transcription and luciferase activity, suggesting a direct TBP-TATA interaction. Importantly, +1 transcription is unaffected by the TATA mutations, suggesting that in the context of pol I recognition, the TATA-containing initiator element serves a function other than TBP binding.

In Arabidopsis thaliana ribosomal RNA genes (rRNA genes or rDNA) are grouped in two nucleolus organizer regions(NORs) that together comprise approximately 6% of the genome. The map positions of the NORs relative to other genetic markers are unknown. It was found that the restriction endonuclease Hindlll cuts once in some but not all rRNA genes to yield strain-specific RFLPs of 100-700 kb that could be visualized by pulsed-field gel electrophoresis and Southern blotting. The Hindlll RFLPs of the A. thaliana strains Columbia and Landsberg segregated among recombinant inbred lines derived from a cross between these two strains. Linkage analysis placed the NOR bearing the polymorphic Hindlll sites to the top of the upper arm of chromosome 2. The name NOR2 is suggested for this locus. Hindlll-bearing rRNA genes ere interspersed with clusters of Hindlll-less genes throughout NOR2. The observed clustering is most consistent with unequal crossing-over, or nearest-neighbor gene conversion, as the mechanism(s) that spread rRNA gene variants throughout an NOR. No meiotic cross-over events yielding a 'hybrid' NOR with multiple RFLPs from both parents were observed among the 47 recombinant inbred lines examined. However,the appearance of novel Hindlll profiles in approximately 40% of the recombinant inbred lines demonstrates that fluctuations in the distribution of rRNA gene variants occur frequently and can be readily detected on pulsed-field gels.

The genes encoding the precursor of 18S, 5.8S and 25S ribosomal RNAs are transcribed in the nucleolus by RNA polymerase I. Unlike rRNA gene promoters in animals which differ substantially across species boundaries, plant rRNA gene promoters share sequence similarity for several nucleotides upstream and downstream of the transcription start site (+1). The conserved sequence consists of a near-consensus TATA box, a critical promoter element of most genes transcribed by RNA polymerase II and certain genes transcribed by RNA polymerase III, followed by a cluster of four to six guanosines. Using transient expression of cloned promoter deletions in Arabidopsis thaliana protoplasts, it is shown that the 5' boundary of the Arabidopsis rRNA gene promoter is located between -55 and -33 and the 3' promoter boundary is approximately +6. The critical role of the TATA element at the initiation site was demonstrated by altering this region using tandem point mutations within constructs containing essentially complete intergenic spacer sequences from -2590 to +6. The initiation site mutations either abolished transcription or dramatically reduced transcript abundance relative to the wild-type promoter. In some mutants, transcription initiation was shifted to a new site, suggesting a role for the TATA-like initiator region in both start site selection and promoter strength. It is suggested that minimal rRNA gene promoters might be similar in design to minimal promoters of protein-encoding genes, many of which utilize an initiator element, or INR, as an important promoter element located directly at the transcription start site.

Arabidopsis thaliana has emerged as a model species for the analysis of genes controlling plant development. However, its small size has impaired biochemical analyses, and the absence of a transient expression system has hampered promoter analysis. Here, we report a method for rapidly establishing A. thaliana suspension cultures that yield protoplasts that can be readily transfected. We have optimized transient expression conditions using a modified polyethylene glycol / calcium nitrate transformation protocol and a Cauliflower Mosaic Virus 35S promoter-luciferase reporter gene construct. Our methods permit isolation of large quantities of rapidly growing cells and analysis of Arabidopsis promoters in vivo in a homologous system.