Respuesta transcripcional de Populus a bifenilos policlorados: Aspectos aplicados a la fitorremediaci��n

La degradaci��n del suelo ha adquirido una magnitud preocupante. Los m��todos tradicionales de descontaminaci��n, son costosos e insuficientes. La fitorremediaci��n representa una alternativa eficaz, de bajo coste, respetuosa con el medio ambiente, que adem��s mejora las propiedades del suelo, si bien ha habido desarrollos relevantes en la ��ltima d��cada. Desde el punto de vida cient��fico, el reto principal es descifrar las rutas metab��licas implicadas en respuesta a contaminantes y comprender su regulaci��n. Esta informaci��n es imprescindible si aspiramos a mejorar las capacidades naturales de algunas especies vegetales para remediar los suelos contaminados. Los estudios de esta Tesis se han centrado en Populus, el mejor modelo forestal disponible a ra��z de la secuenciaci��n de su genoma completo. Por otra parte, Populus tiene una gran capacidad natural para la degradaci��n de contaminantes org��nicos, lo que explica su predominio en los programas forestales de fitorremediaci��n que se desarrollan actualmente. Hemos elegido en concreto al h��brido Populus tremula x P. alba, por la facilidad con que se cultiva y su particular inter��s biotecnol��gico. La presente Tesis plantea un estudio comprehensivo de la respuesta molecular a bifenilos policlorados (PCBs), una familia de contaminantes org��nicos persistentes de particular relevancia a escala mundial. Se ha utilizado para ello una aproximaci��n transcript��mica, basada en tecnolog��a RNA-seq, para identificar los genes implicados en el metabolismo de los compuestos in planta y cuantificar sus niveles de activaci��n en distintas situaciones controladas. La tesis pretende asimismo definir el control transcripcional subyacente a la respuesta bioqu��mica frente a este tipo de contaminantes. Resulta sorprendente que dicha respuesta sea pr��cticamente desconocida a nivel molecular, a pesar de su gran potencial aplicado en el contexto de la tecnolog��a fitorremediadora. Para desarrollar este proyecto aplicamos a nuestros cultivos de chopo h��bridos concentraciones diferentes de Aroclor 1221, una mezcla de PCBs muy utilizada a nivel comercial durante d��cadas, su uso est�� prohibido hoy internacionalmente. Y tomamos muestras de RNA a dos concentraciones y dos momentos distintos de exposici��n al contaminante, generando as�� una matriz de cuatro elementos con sus controles correspondientes. Con el fin de incrementar la especificidad de nuestro an��lisis, consideramos sobre todo los genes diferencialmente expresados m��s significativos seg��n cuatro algoritmos estad��sticos distintos. Por otra parte, realizamos an��lisis funcionales con herramientas bioinform��ticas basadas en comparaciones de secuencias y en redes de co-expresi��n g��nica. La respuesta de los genes de particular inter��s fue validada mediante tecnolog��a qRT-PCR (reacci��n de la polimerasa en cadena cuantitativa en tiempo real). Se trata del primer estudio comprehensivo de la respuesta de un organismo vegetal ante la presencia de PCBs. Este estudio nos ha permitido identificar una cantidad considerable de genes estructurales y reguladores, definiendo nuevos factores de transcripci��n cuya expresi��n es proporcional a la concentraci��n de contaminante en el medio o al tiempo de exposici��n al mismo. Los an��lisis de correlaci��n nos permiten afirmar en que la respuesta metab��lica a PCBs, incluyendo posibles rutas degradadoras, participan en al menos quince factores de transcripci��n y unas cuarenta prote��nas o enzimas que resultan particularmente inducidas. Entre las familias implicadas destacan los citocromos P450, la glutati��n transferasas, las deshidrogenasas reductasas (short-chain dehydrogenase reductase) y las prote��nas MDR (multi-drug resistance). Mientras que los factores de transcripci��n encontrados pertenecen a la familia de ZF-TF, MYBs, WRKYs entre otros. Tambi��n identificamos prote��nas de funci��n desconocida que no se hab��an vinculado previamente a este tipo de respuestas en plantas, como la CSP (cold-shock domain proteins). Para estudiar su posible relaci��n con la presencia de PCBs, se caracteriz�� un gen de esta familia detectado mediante espectrometr��a de masas en t��ndem (MS/MS) a partir de mapas IEF x SDS-PAGE (isoelectro focusing x sodium dodecyl sulphate- polyacrylamide gel electrophoresis) de alta resoluci��n. Mediante qRT-PCR pudimos confirmar la inducci��n del gen correspondiente, ort��logo a PtCSP4 de P. trichocarpa (Potri.004g172600), en respuesta a Aroclor 1221. El an��lisis fenot��pico de las l��neas transg��nicas de Arabidopsis thaliana que sobre-expresaba la prote��na CSP de chopo h��brido confirm�� un papel para la misma tolerancia a PCBs, posiblemente a trav��s de mecanismos reguladores que activan prote��nas MDR. Este trabajo, adem��s de aportar datos novedosos sobre los mecanismos moleculares desencadenados por la presencia de un PCB en Populus, utilizado aqu�� como sistema modelo. Con ello se demuestra el potencial de las especies arb��reas no solo como agentes descontaminantes, ya explotado comercialmente, sino tambi��n como fuente potencial de genes interesantes. Entre los genes identificados en esta Tesis hay candidatos evidentes a participar en mecanismos de tolerancia al estr��s inducido por la contaminaci��n y tambi��n rutas metab��licas degradadores de PCBs. Precisamente la posibilidad de degradar al contaminante confiere particular inter��s a este tipo de estudios frente a la fitorremediaci��n de metales pesados y otros contaminantes elementales. La comparaci��n de los datos generados en este estudio con estudios an��logos que se realicen en el futuro con otras especies y xenobi��ticos, contribuir��n a definir mejor la respuesta de las plantas ante la contaminaci��n org��nica y mejorar su potencial descontaminante. ABSTRACT Soil degradation has acquired a disturbing magnitude. Traditional methods of decontamination are expensive and insufficient. Phytoremediation represent an effective alternative, low cost, respectful of the environment, that also improves soil properties, although there have been relevant developments in the last decade. From a life scientist, the challenge is to decipher the major metabolic pathways involved in response to pollutants and understand their regulation. This information is essential if we desire to enhance the natural abilities of some plant species to remediate contaminated soils. This thesis studies have focused on Populus, the best available forestry model following the sequencing of the entire genome. Moreover, Populus has a natural ability to degrade organic pollutants, which explains its predominance in phytoremediation forestry programs currently being developed. We have chosen specifically to hybrid Populus tremula x P. alba, the ease with which it is grown and its particular biotechnological interest. This thesis presents a comprehensive study of the molecular response to polychlorinated biphenyls (PCBs), a family of persistent organic pollutants of particular relevance worldwide. It has been used for a transcriptomic approach using RNA-seq technology, to identify genes involved in the metabolism of compounds in plant and quantify their levels of activation in different controlled situations. The thesis also aims to define the underlying transcriptional control the biochemical response to these pollutants. It is surprising that the response is virtually unknown at the molecular level, despite its great potential applied in the context of phytoremediation technology. To develop this project we applied our hybrid poplar crops different concentrations of Aroclor 1221, a mixture of PCBs widely used commercially for decades, its use is now banned internationally. And we RNA samples at two different concentrations and times of exposure to the pollutant, generating an array of four elements with their corresponding controls. In order to increase the specificity of our analysis, we consider mainly the most significant differentially expressed genes in four different statistical algorithms. Moreover, functional analyzes conducted with bioinformatics tools based on sequence comparisons and networks gene co-expression. The response of genes of particular interest was validated by qRT-PCR (polymerase reaction chain in real-time quantitative. This is the first comprehensive study of the response of a plant organism in the presence of PCBs. This study allowed us to identify a considerable amount of structural and regulatory genes, defining new transcription factors whose expression is proportional to the concentration of contaminant in the middle or at the time of exposure. Correlation analyzes allow us to affirm that the metabolic response to PCBs, including possible degradative pathways, at least fifteen involved in transcription factors and forty proteins or enzymes which are particularly induced. Among the families involved include cytochromes P450, the glutathione transferases, dehydrogenases reductases (short -chain dehydrogenase reductase) and MDR proteins (multi - drug resistance). While transcription factors belong to the family found ZF-TF, MYBs, WRKYs among others. We also identify proteins of unknown function that had not been previously linked to such responses in plants such as CSP (cold- shock domain proteins). To study their possible relationship with the presence of PCBs, a gene in this family was characterized and was detected by tandem mass spectrometry (MS/MS) from maps IEF x SDS -PAGE (sodium dodecyl isoelectro x sulphate- polyacrylamide gel electrophoresis) of high resolution. By qRT -PCR could confirm the induction of the corresponding gene, ortholog to PtCSP4 of P. trichocarpa (Potri.004g172600), in response to Aroclor 1221. Phenotypic analysis of transgenic Arabidopsis thaliana lines over- expressing the protein CSP poplar hybrid confirmed a role for PCBs same tolerance, possibly through regulatory mechanisms activated MDR proteins. This work, in addition to providing new data on the molecular mechanisms triggered by the presence of PCBs in Populus, used here as a model system. Thus the potential of tree species not only as decontamination agents, and commercially exploited, but also as a potential source of interesting genes is shown. Among the genes identified in this thesis there are evident candidates to participate in tolerance mechanisms to stress induced by pollution and degrading metabolic pathways of PCBs. Precisely the possibility of degrading the pollutant attaches particular interest to this type of study off the phytoremediation of heavy metals and other elemental pollutants. The comparison of the data generated in this study with similar studies carried out in the future with other species and xenobiotics contribute to better define the response of plants to organic pollution and improve their decontamination potential.

Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis

Vitamin C (l-ascorbic acid; AsA) acts as a potent antioxidant and cellular reductant in plants and animals. AsA has long been known to have many critical physiological roles in plants, yet its biosynthesis is only currently being defined. A pathway for AsA biosynthesis that features GDP-mannose and l-galactose has recently been proposed for plants. We have isolated a collection of AsA-deficient mutants of Arabidopsis thaliana that are valuable tools for testing of an AsA biosynthetic pathway. The best-characterized of these mutants (vtc1) contains ���25% of wild-type AsA and is defective in AsA biosynthesis. By using a combination of biochemical, molecular, and genetic techniques, we have demonstrated that the VTC1 locus encodes a GDP-mannose pyrophosphorylase (mannose-1-P guanyltransferase). This enzyme provides GDP-mannose, which is used for cell wall carbohydrate biosynthesis and protein glycosylation as well as for AsA biosynthesis. In addition to genetically defining the first locus involved in AsA biosynthesis, this work highlights the power of using traditional mutagenesis techniques coupled with the Arabidopsis Genome Initiative to rapidly clone physiologically important genes.

Agrobacterium VirD2 protein interacts with plant host cyclophilins

Agrobacterium tumefaciens induces crown gall tumors on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of T-DNA, a single-stranded DNA segment of the tumor-inducing plasmid, VirD2, an endonuclease covalently bound to the 5��� end of the T-DNA, and perhaps VirE2, a single-stranded DNA binding protein. The yeast two-hybrid system was used to screen for proteins interacting with VirD2 and VirE2 to identify components in Arabidopsis thaliana that interact with the T-complex. Three VirD2- and two VirE2-interacting proteins were identified. Here we characterize the interactions of VirD2 with two isoforms of Arabidopsis cyclophilins identified by using this analysis. The VirD2 domain interacting with the cyclophilins is distinct from the endonuclease, omega, and the nuclear localization signal domains. The VirD2���cyclophilin interaction is disrupted in vitro by cyclosporin A, which also inhibits Agrobacterium-mediated transformation of Arabidopsis and tobacco. These data strongly suggest that host cyclophilins play a role in T-DNA transfer.

Cloning and characterization of a plastidal and a mitochondrial isoform of tobacco protoporphyrinogen IX���oxidase

Protoporphyrinogen IX oxidase is the last enzyme in the common pathway of heme and chlorophyll synthesis and provides precursor for the mitochondrial and plastidic heme synthesis and the predominant chlorophyll synthesis in plastids. We cloned two different, full-length tobacco cDNA sequences by complementation of the protoporphyrin-IX-accumulating Escherichia coli hemG mutant from heme auxotrophy. The two sequences show similarity to the recently published Arabidopsis PPOX, Bacillus subtilis hemY, and to mammalian sequences encoding protoporphyrinogen IX oxidase. One cDNA sequence encodes a 548-amino acid residues protein with a putative transit sequence of 50 amino acid residues, and the second cDNA encodes a protein of 504 amino acid residues. Both deduced protein sequences share 27.2% identical amino acid residues. The first in vitro translated protoporphyrinogen IX oxidase could be translocated to plastids, and the approximately 53-kDa mature protein was detected in stroma and membrane fraction. The second enzyme was targeted to mitochondria without any detectable reduction in size. Localization of both enzymes in subcellular fractions was immunologically confirmed. Steady-state RNA analysis indicates an almost synchronous expression of both genes during tobacco plant development, greening of young seedlings, and diurnal and circadian growth. The mature plastidal and the mitochondrial isoenzyme were overexpressed in E. coli. Bacterial extracts containing the recombinant mitochondrial enzyme exhibit high protoporphyrinogen IX oxidase activity relative to control strains, whereas the plastidal enzyme could only be expressed as an inactive peptide. The data presented confirm a compartmentalized pathway of tetrapyrrole synthesis with protoporphyrinogen IX oxidase in plastids and mitochondria.

Plant ionomics : From elemental profiling to environmental adaptation

FUNDING UK Biotechnology and Biological Sciences Research Council grant BB/L027739/1 and BB/L000113/1 (to D.E.S.), the US National Institutes of Health grant 2R01GM078536 (to D.E.S.), and the US National Science Foundation grant IOB 0419695 (to D.E.S.) ACKNOWLEDGMENTS We wish to thank our collaborators Mary Lou Guerinot, Niko Geldner, and Christian Hermans for kindly allowing us to incorporate in this update unpublished data on BRUTUS, SGN1, and SGN3, respectively. We also thank Mary Lou Guerinot, Niko Geldner, Takehiro Kamiya, and the ERACAPS Root Barrier project for productive discussions relating to ionomics and the plant ionome. No conflict of interest declared.

Dinor-oxo-phytodienoic acid: A new hexadecanoid signal in the jasmonate���family

Jasmonic acid and its precursors are potent regulatory molecules in plants. We devised a method for the simultaneous extraction of these compounds from plant leaves to quantitate changes in the levels of jasmonate family members during health and on wounding. During our study, we identified a novel 16-carbon cyclopentenoic acid in leaf extracts from Arabidopsis and potato. The new compound, a member of the jasmonate family of signals, was named dinor-oxo-phytodienoic acid. Dinor-oxo-phytodienoic acid was not detected in the Arabidopsis mutant fad5, which is incapable of synthesizing 7Z,10Z,13Z-hexadecatrienoic acid (16:3), suggesting that the metabolite is derived directly from plastid 16:3 rather than by ��-oxidation of the 18-carbon 12-oxo-phytodienoic acid. Simultaneous quantitation of jasmonate family members in healthy leaves of Arabidopsis and potato suggest that different plant species have different relative levels of jasmonic acid, oxo-phytodienoic acid, and dinor-oxo-phytodienoic acid. We term these profiles ���oxylipin signatures.��� Dinor-oxo-phytodienoic acid levels increased dramatically in Arabidopsis and potato leaves on wounding, suggesting roles in wound signaling. Treatment of Arabidopsis with micromolar levels of dinor-oxo-phytodienoic acid increased the ability of leaf extracts to transform linoleic acid into the ��-ketol 13-hydroxy-12-oxo-9(Z) octadecenoic acid indicating that the compound can regulate part of its own biosynthetic pathway. Tightly regulated changes in the relative levels of biologically active jasmonates may permit sensitive control over metabolic, developmental, and defensive processes in plants.

Protein���protein interactions among the Aux/IAA���proteins

The plant hormone indoleacetic acid (IAA) transcriptionally activates early genes in plants. The Aux/IAA family of early genes encodes proteins that are short-lived and nuclear-localized. They also contain a putative prokaryotic ������ DNA binding motif whose formation requires protein dimerization. Here, we show that the pea PS-IAA4 and Arabidopsis IAA1 and IAA2 proteins perform homo- and heterotypic interactions in yeast using the two-hybrid system. Gel-filtration chromatography and chemical cross-linking experiments demonstrate that the PS-IAA4 and IAA1 proteins interact to form homodimers in vitro. Deletion analysis of PS-IAA4 indicates that the ������ containing acidic C terminus of the protein is necessary for homotypic interactions in the yeast two-hybrid system. Screening an Arabidopsis ��-ACT cDNA library using IAA1 as a bait reveals heterotypic interactions of IAA1 with known and newly discovered members of the Arabidopsis Aux/IAA gene family. The new member IAA24 has similarity to ARF1, a transcription factor that binds to an auxin response element. Combinatorial interactions among the various members of the Aux/IAA gene family may regulate a variety of late genes as well as serve as autoregulators of early auxin-regulated gene expression. These interactions provide a molecular basis for the developmental and tissue-specific manner of auxin action.

Gene discovery in the wood-forming tissues of poplar: Analysis of 5,692 expressed sequence tags

A rapidly growing area of genome research is the generation of expressed sequence tags (ESTs) in which large numbers of randomly selected cDNA clones are partially sequenced. The collection of ESTs reflects the level and complexity of gene expression in the sampled tissue. To date, the majority of plant ESTs are from nonwoody plants such as Arabidopsis, Brassica, maize, and rice. Here, we present a large-scale production of ESTs from the wood-forming tissues of two poplars, Populus tremula L. �� tremuloides Michx. and Populus trichocarpa ���Trichobel.��� The 5,692 ESTs analyzed represented a total of 3,719 unique transcripts for the two cDNA libraries. Putative functions could be assigned to 2,245 of these transcripts that corresponded to 820 protein functions. Of specific interest to forest biotechnology are the 4% of ESTs involved in various processes of cell wall formation, such as lignin and cellulose synthesis, 5% similar to developmental regulators and members of known signal transduction pathways, and 2% involved in hormone biosynthesis. An additional 12% of the ESTs showed no significant similarity to any other DNA or protein sequences in existing databases. The absence of these sequences from public databases may indicate a specific role for these proteins in wood formation. The cDNA libraries and the accompanying database are valuable resources for forest research directed toward understanding the genetic control of wood formation and future endeavors to modify wood and fiber properties for industrial use.

A reversibly glycosylated polypeptide (RGP1) possibly involved in plant cell wall synthesis: Purification, gene cloning, and trans-Golgi���localization

We purified from pea (Pisum sativum) tissue an ���40 kDa reversibly glycosylated polypeptide (RGP1) that can be glycosylated by UDP-Glc, UDP-Xyl, or UDP-Gal, and isolated a cDNA encoding it, apparently derived from a single-copy gene (Rgp1). Its predicted translation product has 364 aminoacyl residues and molecular mass of 41.5 kDa. RGP1 appears to be a membrane-peripheral protein. Immunogold labeling localizes it specifically to trans-Golgi dictyosomal cisternae. Along with other evidence, this suggests that RGP1 is involved in synthesis of xyloglucan and possibly other hemicelluloses. Corn (Zea mays) contains a biochemically similar and structurally homologous RGP1, which has been thought (it now seems mistakenly) to function in starch synthesis. The expressed sequence database also reveals close homologs of pea Rgp1 in Arabidopsis and rice (Oryza sativa). Rice possesses, in addition, a distinct but homologous sequence (Rgp2). RGP1 provides a polypeptide marker for Golgi membranes that should be useful in plant membrane studies.

Crystal structure of SQD1, an enzyme involved in the biosynthesis of the plant sulfolipid headgroup donor UDP-sulfoquinovose

The SQD1 enzyme is believed to be involved in the biosynthesis of the sulfoquinovosyl headgroup of plant sulfolipids, catalyzing the transfer of SO3��� to UDP-glucose. We have determined the structure of the complex of SQD1 from Arabidopsis thaliana with NAD+ and the putative substrate UDP-glucose at 1.6-��� resolution. Both bound ligands are completely buried within the binding cleft, along with an internal solvent cavity which is the likely binding site for the, as yet, unidentified sulfur-donor substrate. SQD1 is a member of the short-chain dehydrogenase/reductase (SDR) family of enzymes, and its structure shows a conservation of the SDR catalytic residues. Among several highly conserved catalytic residues, Thr-145 forms unusually short hydrogen bonds with both susceptible hydroxyls of UDP-glucose. A His side chain may also be catalytically important in the sulfonation.

Stimulation of the blue light phototropic receptor NPH1 causes a transient increase in cytosolic Ca2+

Blue light regulates plant growth and development, and three photoreceptors, CRY1, CRY2, and NPH1, have been identified. The transduction pathways of these receptors are poorly understood. Transgenic plants containing aequorin have been used to dissect the involvement of these three receptors in the regulation of intracellular Ca2+. Pulses of blue light induce cytosolic Ca2+ transients lasting about 80 s in Arabidopsis and tobacco seedlings. Use of organelle-targeted aequorins shows that Ca2+ increases are limited to the cytoplasm. Blue light treatment of cry1, cry2, and nph1 mutants showed that NPH1, which regulates phototropism, is largely responsible for the Ca2+ transient. The spectral response of the Ca2+ transient is similar to that of phototropism, supporting NPH1 involvement. Furthermore, known interactions between red and blue light and between successive blue light pulses on phototropic sensitivity are mirrored in the blue light control of cytosolic Ca2+ in these seedlings. Our observations raise the possibility that physiological responses regulated by NPH1, such as phototropism, may be transduced through cytosolic Ca2+.

Sulfate reduction in higher plants: Molecular evidence for a novel 5���-adenylylsulfate���reductase

Sulfate-assimilating organisms reduce inorganic sulfate for Cys biosynthesis. There are two leading hypotheses for the mechanism of sulfate reduction in higher plants. In one, adenosine 5���-phosphosulfate (APS) (5���-adenylylsulfate) sulfotransferase carries out reductive transfer of sulfate from APS to reduced glutathione. Alternatively, the mechanism may be similar to that in bacteria in which the enzyme, 3���-phosphoadenosine-5���-phosphosulfate (PAPS) reductase, catalyzes thioredoxin (Trx)-dependent reduction of PAPS. Three classes of cDNA were cloned from Arabidopsis thaliana termed APR1, -2, and -3, that functionally complement a cysH, PAPS reductase mutant strain of Escherichia coli. The coding sequence of the APR clones is homologous with PAPS reductases from microorganisms. In addition, a carboxyl-terminal domain is homologous with members of the Trx superfamily. Further genetic analysis showed that the APR clones can functionally complement a mutant strain of E. coli lacking Trx, and an APS kinase, cysC. mutant. These results suggest that the APR enzyme may be a Trx-independent APS reductase. Cell extracts of E. coli expressing APR showed Trx-independent sulfonucleotide reductase activity with a preference for APS over PAPS as a substrate. APR-mediated APS reduction is dependent on dithiothreitol, has a pH optimum of 8.5, is stimulated by high ionic strength, and is sensitive to inactivation by 5���-adenosinemonophosphate (5���-AMP). 2���-AMP, or 3���-phosphoadenosine-5���-phosphate (PAP), a competitive inhibitor of PAPS reductase, do not affect activity. The APR enzymes may be localized in different cellular compartments as evidenced by the presence of an amino-terminal transit peptide for plastid localization in APR1 and APR3 but not APR2. Southern blot analysis confirmed that the APR clones are members of a small gene family, possibly consisting of three members.

In silico detection of control signals: mRNA 3���-end-processing sequences in diverse species

We have investigated mRNA 3���-end-processing signals in each of six eukaryotic species (yeast, rice, arabidopsis, fruitfly, mouse, and human) through the analysis of more than 20,000 3���-expressed sequence tags. The use and conservation of the canonical AAUAAA element vary widely among the six species and are especially weak in plants and yeast. Even in the animal species, the AAUAAA signal does not appear to be as universal as indicated by previous studies. The abundance of single-base variants of AAUAAA correlates with their measured processing efficiencies. As found previously, the plant polyadenylation signals are more similar to those of yeast than to those of animals, with both common content and arrangement of the signal elements. In all species examined, the complete polyadenylation signal appears to consist of an aggregate of multiple elements. In light of these and previous results, we present a broadened concept of 3���-end-processing signals in which no single exact sequence element is universally required for processing. Rather, the total efficiency is a function of all elements and, importantly, an inefficient word in one element can be compensated for by strong words in other elements. These complex patterns indicate that effective tools to identify 3���-end-processing signals will require more than consensus sequence identification.

A functional homolog of a yeast tRNA splicing enzyme is conserved in higher eukaryotes and in Escherichia coli

tRNA splicing in the yeast Saccharomyces cerevisiae requires an endonuclease to excise the intron, tRNA ligase to join the tRNA half-molecules, and 2���-phosphotransferase to transfer the splice junction 2���-phosphate from ligated tRNA to NAD, producing ADP ribose 1���������2������ cyclic phosphate (Appr>p). We show here that functional 2���-phosphotransferases are found throughout eukaryotes, occurring in two widely divergent yeasts (Candida albicans and Schizosaccharomyces pombe), a plant (Arabidopsis thaliana), and mammals (Mus musculus); this finding is consistent with a role for the enzyme, acting in concert with ligase, to splice tRNA or other RNA molecules. Surprisingly, functional 2���-phosphotransferase is found also in the bacterium Escherichia coli, which does not have any known introns of this class, and does not appear to have a ligase that generates junctions with a 2���-phosphate. Analysis of the database shows that likely members of the 2���-phosphotransferase family are found also in one other bacterium (Pseudomonas aeruginosa) and two archaeal species (Archaeoglobus fulgidus and Pyrococcus horikoshii). Phylogenetic analysis reveals no evidence for recent horizontal transfer of the 2���-phosphotransferase into Eubacteria, suggesting that the 2���-phosphotransferase has been present there since close to the time that the three kingdoms diverged. Although 2���-phosphotransferase is not present in all Eubacteria, and a gene disruption experiment demonstrates that the protein is not essential in E. coli, the continued presence of 2���-phosphotransferase in Eubacteria over large evolutionary times argues for an important role for the protein.

The circadian clock controls the expression pattern of the circadian input photoreceptor, phytochrome B

Developmental and physiological responses are regulated by light throughout the entire life cycle of higher plants. To sense changes in the light environment, plants have developed various photoreceptors, including the red/far-red light-absorbing phytochromes and blue light-absorbing cryptochromes. A wide variety of physiological responses, including most light responses, also are modulated by circadian rhythms that are generated by an endogenous oscillator, the circadian clock. To provide information on local time, circadian clocks are synchronized and entrained by environmental time cues, of which light is among the most important. Light-driven entrainment of the Arabidopsis circadian clock has been shown to be mediated by phytochrome A (phyA), phytochrome B (phyB), and cryptochromes 1 and 2, thus affirming the roles of these photoreceptors as input regulators to the plant circadian clock. Here we show that the expression of PHYB���LUC reporter genes containing the promoter and 5��� untranslated region of the tobacco NtPHYB1 or Arabidopsis AtPHYB genes fused to the luciferase (LUC) gene exhibit robust circadian oscillations in transgenic plants. We demonstrate that the abundance of PHYB RNA retains this circadian regulation and use a PHYB���Luc fusion protein to show that the rate of PHYB synthesis is also rhythmic. The abundance of bulk PHYB protein, however, exhibits only weak circadian rhythmicity, if any. These data suggest that photoreceptor gene expression patterns may be significant in the daily regulation of plant physiology and indicate an unexpectedly intimate relationship between the components of the input pathway and the putative circadian clock mechanism in higher plants.