The ubiquitous endonuclease RNase P is responsible for the 5' maturation of tRNA precursors. Until the discovery of human mitochondrial RNase P, these enzymes had typically been found to be ribonucleoproteins, the catalytic activity of which is associated with the RNA component. Here we show that, in Arabidopsis thaliana mitochondria and plastids, a single protein called 'proteinaceous RNase P' (PRORP1) can perform the endonucleolytic maturation of tRNA precursors that defines RNase P activity. In addition, PRORP1 is able to cleave tRNA-like structures involved in the maturation of plant mitochondrial mRNAs. Finally, we show that Arabidopsis PRORP1 can replace the bacterial ribonucleoprotein RNase P in Escherichia coli cells. PRORP2 and PRORP3, two paralogs of PRORP1, are both localized in the nucleus.

Manganese toxicity is a major problem for plant growth in acidic soils, but cellular mechanisms that facilitate growth in such conditions have not been clearly delineated. Established mechanisms that counter metal toxicity in plants involve chelation and cytoplasmic export of the metal across the plasma or vacuolar membranes out of the cell or sequestered into a large organelle, respectively. We report here that expression of the Arabidopsis and poplar MTP11 cation diffusion facilitators in a manganesehypersensitive yeast mutant restores manganese tolerance to wild-type levels. Microsomes from yeast expressing AtMTP11 exhibit enhanced manganese uptake. In accord with a presumed function of MTP11 in manganese tolerance, Arabidopsis mtp11 mutants are hypersensitive to elevated levels of manganese, whereas plants overexpressing MTP11 are hypertolerant. In contrast, sensitivity to manganese deficiency is slightly decreased in mutants and increased in overexpressing lines. Promoter-GUS studies showed that AtMTP11 is most highly expressed in root tips, shoot margins, and hydathodes, but not in epidermal cells and trichomes, which are generally associated with manganese accumulation. Surprisingly, imaging of MTP11-EYFP fusions demonstrated that MTP11 localizes neither to the plasma membrane nor to the vacuole, but to a punctate endomembrane compartment that largely coincides with the distribution of the trans-Golgi marker sialyl transferase. Golgi-based manganese accumulation might therefore result in manganese tolerance through vesicular trafficking and exocytosis. In accord with this proposal, Arabidopsis mtp11 mutants exhibit enhanced manganese concentrations in shoots and roots. We propose that Golgi-mediated exocytosis comprises a conserved mechanism for heavy metal tolerance in plants.Golgi ͉ heavy metal transport ͉ metal tolerance protein ͉ metal trafficking ͉ manganese transporter T ransition metals are required by living systems where they perform a wide variety of functions as cofactors for enzymes and transcription factors. Transition metals are also present in many environments at potentially toxic concentrations, and this has led to the evolution of mechanisms that counter toxicity. In plants exposed to high concentrations of transition metals in the soil, binding of the metals to phytochelatins in the cytosol lowers metal activity (1). Additionally, metals can be removed from the cytosol through the action of metal transporters. Transporters involved in metal tolerance localize to the plasma membrane, thereby removing metals from the cell, or to the vacuolar membrane, where the metal can be sequestered into a large and metabolically relatively inert intracellular compartment (2).Manganese is the second most prevalent transition metal, after iron, in the Earth's crust and an essential micronutrient for all organisms, including humans and plants (3). In addition to being a cofactor for a variety of enzymes (including various decarboxylases of the tricarboxylic acid cycle, RNA polymerases, and numerous glyco...

The Arabidopsis thaliana genome contains five genes that encode two pore K ؉ (TPK) channels. The most abundantly expressed isoform of this family, TPK1, is expressed at the tonoplast where it mediates K ؉ -selective currents between cytoplasmic and vacuolar compartments. TPK1 open probability depends on both cytoplasmic Ca 2؉ and cytoplasmic pH but not on the tonoplast membrane voltage. The channel shows intrinsic rectification and can be blocked by Ba 2؉ , tetraethylammonium, and quinine. TPK1 current was found in all shoot cell types and shows all of the hallmarks of the previously described vacuolar K (VK) tonoplast channel characterized in guard cells. Characterization of TPK1 loss-of-function mutants and TPK1-overexpressing plants shows that TPK1 has a role in intracellular K ؉ homeostasis affecting seedling growth at high and low ambient K ؉ levels. In stomata, TPK1 function is consistent with vacuolar K ؉ release, and removal of this channel leads to slower stomatal closure kinetics. During germination, TPK1 contributes to the radicle development through vacuolar K ؉ deposition to provide expansion growth or in the redistribution of essential minerals.

RNase P is an essential enzyme that cleaves the 59 leader sequence of tRNA precursors. RNase Ps were believed until now to occur universally as ribonucleoproteins in organisms performing RNase P activity. Here we find that protein-only RNase P enzymes called PRORP (for proteinaceous RNase P) support RNase P activity in vivo in both organelles and the nucleus in Arabidopsis. Beyond tRNA, PRORP proteins are involved in the maturation of small nucleolar RNA (snoRNA) and mRNA. Finally, ribonucleoprotein RNase MRP is not involved in tRNA maturation in plants. Altogether, our results indicate that ribonucleoprotein enzymes have been entirely replaced by proteins for RNase P activity in plants.Supplemental material is available for this article.Received February 10, 2012; revised version accepted April 5, 2012.RNase P is a virtually universal enzyme involved in the maturation of tRNAs, as it cleaves the 59 leader sequence of tRNA precursors. It is thus essential to obtain functional tRNAs and is therefore pivotal for translation (Lai et al. 2010;Reiter et al. 2010). RNase P activities from all phyla of life were assumed to be universally performed by ribonucleoprotein enzymes whose catalytic activities are held by ribozymes (Altman 2007). This concept was first challenged with the proposition that spinach chloroplast and human mitochondria RNase Ps would not contain any RNA moiety (Wang et al. 1988;Rossmanith and Karwan 1998). More recently, protein-only RNase P enzymes called PRORP (for proteinaceous RNase P) have been characterized at the molecular level in endosymbiotic organelles in both humans and Arabidopsis (Holzmann et al. 2008;Gobert et al. 2010). Still, the dogma remained that RNase P enzymes would nonetheless universally occur as ribonucleoproteins in living organisms performing RNase P activity, with protein-only RNase Ps being marginal exceptions restricted to only some organelles (e.g., Esakova and Krasilnikov 2010).The putative PRORP RNase P enzymes are characterized by the occurrence of a conserved ''NYN'' metallonuclease domain (Anantharaman and Aravind 2006). PRORP proteins also belong to the huge pentatricopeptide repeat (PPR) protein family. These proteins, typically from eukaryotes, are involved in a wide variety of posttranscriptional mechanisms (Schmitz-Linneweber and Small 2008). However, no functional information was available for PRORP proteins. We previously established that Arabidopsis PRORP1, a protein localized in organelles, can act in vitro as an RNase P enzyme that is a single protein (Gobert et al. 2010), although its function remained elusive in planta. We also used localization experiments (YFP fusions and immunodetections) to determine that PRORP2 and PRORP3, two paralogs of PRORP1, were present in Arabidopsis nuclei (Gobert et al. 2010). In addition, the fast-growing amount of genomic data has revealed that some important groups of eukaryotes, such as land plants and kinetoplastids, do not encode any recognizable genes for RNase P RNA or for proteins specific for ribonucleoprotein...

The Arabidopsis thaliana genome contains 20 cyclic nucleotide gated channel (CNGC) genes encoding putative non-selective ion channels. Classical and reverse genetic approaches have revealed that two members of this family (CNGC2 and CNGC4) play a role in plant defence responses whereas CNGC1 and CNGC10 may participate in heavy metal and cation transport. Yet, it remains to be resolved how the ion transport attributes of CNGCs are integrated into their physiological function. In this study, CNGC3 is characterized through heterologous expression, GUS- and GFP-reporter gene fusions, and by adopting a reverse genetics approach. A CNGC3-GFP fusion protein shows that it is mainly targeted to the plasma membrane. Promoter GUS studies demonstrate CNGC3 expression predominantly in the cortical and epidermal root cells, but also a ubiquitous presence in shoot tissues. Expression of CNGC3 in yeast indicates it can function as a Na(+) uptake and a K(+) uptake mechanism. cngc3 null mutations decreased seed germination in the presence of NaCl but not KCl. Relative to the wild type, mutant seedling growth is more resistant to the presence of toxic concentrations of NaCl and KCl. The ionic composition and ion uptake characteristics of wild-type and mutant seedlings suggests that the growth advantage in these conditions may be due to restricted ion influx in mutant plants, and that CNGC3 functions in the non-selective uptake of monovalent cations in Arabidopsis root tissue.

RNase P is the essential activity removing 5′-leader sequences from transfer RNA precursors. RNase P was always associated with ribonucleoprotein complexes before the discovery of protein-only RNase P enzymes called PRORPs (PROteinaceous RNase P) in eukaryotes. Here we provide biophysical and functional data to understand the mode of action of PRORP enzymes. Activity assays and footprinting experiments show that the anticodon domain of transfer RNA is dispensable, whereas individual residues in D and TψC loops are essential for PRORP function. PRORP proteins are characterized in solution and a molecular envelope is derived from small-angle X-ray scattering. Conserved residues are shown to be involved in the binding of one zinc atom to PRORP. These results facilitate the elaboration of a model of the PRORP/transfer RNA interaction. The comparison with the ribonucleoprotein RNase P/transfer RNA complex suggests that transfer RNA recognition by PRORP proteins is similar to that by ribonucleoprotein RNase P.

Following the endosymbiotic acquisition of mitochondria by eukaryotic cells, most of the genes in this organelle were transferred to the nucleus. To maintain mitochondrial biogenesis and function, nuclear and mitochondrial genomes require regulated and coordinated expression. In plant organelles, nuclear-encoded proteins targeted to the organelles control posttranscriptional and posttranslational mechanisms. Pentatricopeptide repeat (PPR) proteins are good candidates to play such regulatory roles. Here, we identify PNM1 (for PPR protein localized to the nucleus and mitochondria 1), a novel PPR protein that is dual localized to mitochondria and nuclei in Arabidopsis thaliana, as observed by green fluorescent protein fusions and immunodetection on subcellular fractions and on histological sections. Genetic complementation showed that loss of PNM1 function in mitochondria, but not in nuclei, is lethal for the embryo. In mitochondria, it is associated with polysomes and may play a role in translation. A genetic screen in yeast identified protein partners of PNM1. These partners, the nucleosome assembly protein NAP1, and the transcription factor TCP8 interact with PNM1 in the nucleus in planta. Furthermore, TCP8 can bind the promoter of PNM1. This suggests that PNM1 might be involved in the regulation of its own gene expression in the nucleus and could thus play a role in gene expression adjustments between mitochondria and the nucleus.

RNase P is the endonuclease that removes 5' leader sequences from tRNA precursors. In Eukarya, separate RNase P activities exist in the nucleus and mitochondria/plastids. Although all RNase P enzymes catalyze the same reaction, the different architectures found in Eukarya range from ribonucleoprotein (RNP) enzymes with a catalytic RNA and up to 10 protein subunits to single-subunit protein-only RNase P (PRORP) enzymes. Here, analysis of the phylogenetic distribution of RNP and PRORP enzymes in Eukarya revealed 1) a wealth of novel P RNAs in previously unexplored phylogenetic branches and 2) that PRORP enzymes are more widespread than previously appreciated, found in four of the five eukaryal supergroups, in the nuclei and/or organelles. Intriguingly, the occurrence of RNP RNase P and PRORP seems mutually exclusive in genetic compartments of modern Eukarya. Our comparative analysis provides a global picture of the evolution and diversification of RNase P throughout Eukarya.