Evidence in support of biosynthesis de novo of free cytokinins

The four cytokinins in the tRNA from Lupinus luteus L. seeds have been purified and identified as ribosyl-cis-zeatin, 2-methylthio-ribosylzeatin, ( ²-isopentenyl)adenosine and 2-methylthio-N⁶-( ²-isopentenyl)adenosine. These structures have been assigned on the basis of their chromatographic mobilities and the spectroscopic data of the parent materials and their silylated derivatives. The tRNA isolated from Populus x robusta Schneid. leaves contained four cytokinins with identical chromatographic properties to those identified in Lupinus luteus seed tRNA. No evidence was obtained for the presence, in tRNA, of the naturally occurring free cytokinins identified in these plant species, dihydrozeatin (Lupinus luteus) and N⁶-(2-hydroxybenzyl)adenosine (Populus x robusta). This is evidence in support of the possibility that free cytokinins can arise by biosynthesis de novo and are not exclusively by-products released intact during tRNA turnover.     

Transgenic grapefruit plants expressing the PAPETALA3-IPT gp gene exhibit altered expression of PR genes

Transgenic grapefruit plants (Citrus paradisi cv. ‘Duncan’) with the isopentenyltransferase (ipt) gene under the control of APETALA3 promoter have been produced using Agrobacterium-mediated transformation. The relative expression level of the ipt gene was between 2.3 and 7 times higher in transformed plants than in the wild-type but despite the presence of a tissue-specific promoter, the expression was not limited only to flower tissue. Increased levels of trans-zeatin riboside between 9.4 and 32-fold found in transgenic grapefruit were considered the consequence of ectopic expression of the ipt gene. Chlorophyll levels in fully expanded uppermost leaves were also about 30% higher in transgenic than in wild-type plants. Involvement of cytokinins in control of expression of three pathogenesis-related protein genes: β-1,3-glucanase, a stress related PR gene 24P220, and an acidic chitinase, 24P262 was examined. Expression of β-1,3-glucanase, and 24P220 gene were significantly enhanced in transgenic plants while the expression of chitinase was reduced to low levels. Our results confirm the effect of cytokinins on expression of genes implicated in the response of grapefruit plants to pathogen attack and suggest a possible role of cytokinins in pathogen resistance.     

Gamma-irradiated plant callus tissue: Cytokinins from transfer ribonucleic acid

Summary Callus tissue ofHaworthia mirabilis Haw. was irradiated with⁶⁰Co gamma rays. tRNA was isolated, hydrolyzed enzymatically, and cytokinin-active ribonucleosides were separated by Sephadex LH-20 column chromatography and assayed with the tobaccocallus cytokinin bioassay. Three cytokinins were detected in tRNA from irradiated tissue, two of which chromatographed with zeatin riboside and N⁶-(Δ²-isopentenyl)adenosine. The third cytokinin-active ribonucleoside was retained longer than the above compounds on the Sephadex column and may be 2-methylthio-N⁶-(Δ²-isopentenyl)adenosine. Two cytokinins were detected in tRNA from nonirradiated tissue—those chromatographed with zeatin riboside and N⁶-(Δ²-isopentenyl)adenosine. Relationships between cytokinins from tRNA and free cytokinins found in tissue earlier are discussed. This is paper 78-10-124 of the Kentucky Agricultural Experiment Staton and is published with approval of the Director.     

Evolution of +1 Programmed Frameshifting Signals and Frameshift-Regulating tRNAs in the Order Saccharomycetales

Programmed translational frameshifting is a ubiquitous but rare mechanism of gene expression in which mRNA sequences cause the translational machinery to shift reading frames with extreme efficiency, up to at least 50%. The mRNA sequences responsible are deceptively simple; the sequence CUU-AGG-C causes about 40% frameshifting when inserted into an mRNA in the yeast Saccharomyces cerevisiae. The high efficiency of this site depends on a set of S. cerevisiae tRNA isoacceptors that perturb the mechanism of translation to cause the programmed translational error. The simplicity of the system might suggest that it could evolve frequently and perhaps be lost as easily. We have investigated the history of programmed +1 frameshifting in fungi. We find that frameshifting has persisted in two structural genes in budding yeasts, ABP140 and EST3 for about 150 million years. Further, the tRNAs that stimulate the event are equally old. Species that diverged from the lineage earlier both do not employ frameshifting and have a different complement of tRNAs predicted to be inimical to frameshifting. The stability of the coevolution of protein coding genes and tRNAs suggests that frameshifting has been selected for during the divergence of these species. [Reviewing Editor: Dr. Niles Lehman]     

Schizosaccharomyces pombe contains separate CC- and A-adding tRNA nucleotidyltransferases

A specific cytidine-cytidine-adenosine (CCA) sequence is required at the 3′-terminus of all functional tRNAs. This sequence is added during tRNA maturation or repair by tRNA nucleotidyltransferase enzymes. While most eukaryotes have a single enzyme responsible for CCA addition, some bacteria have separate CC- and A-adding activities. The fungus, Schizosaccharomyces pombe, has two genes (cca1 and cca2) that are thought, based on predicted amino acid sequences, to encode tRNA nucleotidyltransferases. Here, we show that both genes together are required to complement a Saccharomyces cerevisiae strain bearing a null mutation in the single gene encoding its tRNA nucleotidyltransferase. Using enzyme assays we show further that the purified S. pombe cca1 gene product specifically adds two cytidine residues to a tRNA substrate lacking this sequence while the cca2 gene product specifically adds the terminal adenosine residue thereby completing the CCA sequence. These data indicate that S. pombe represents the first eukaryote known to have separate CC- and A-adding activities for tRNA maturation and repair. In addition, we propose that a novel structural change in a tRNA nucleotidyltransferase is responsible for defining a CC-adding enzyme.     

Calcium regulates the expression of a<Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> asparaginyl tRNA synthetase gene

In a screen for calcium-regulated gene expression during growth and development ofDictyostelium discoideum we have identified an asparaginyl tRNA synthetase (ddAsnRS) gene, the second tRNA synthetase gene identified in this organism. TheddAsnRS gene shows many unique features. One, it is repressed by lowering cellular calcium, making it the first known calcium-regulated tRNA synthetase. Two, despite the calcium-dependence, its expression is unaltered during the cell cycle, making this the firstD. discoideum gene to show a calcium-dependent but cell cycle phase-independent expression. Finally, the N-terminal domain of the predicted ddAsnRS protein shows higher sequence similarity to Glutaminyl tRNA synthetases than to other Asn tRNA synthetases. These unique features of theAsnRS from this primitive eukaryote not only point to a novel mechanism regulating the components of translation machinery and gene expression by calcium, but also hint at a link between the evolution ofGlnRS andAsnRS in eukaryotes.     

The Discriminatory Transfer Routes of tRNA Genes Among Organellar and Nuclear Genomes in Flowering Plants: A Genome-Wide Investigation of <Emphasis Type="Italic">indica</Emphasis> Rice

The transfer and integration of tRNA genes from organellar genomes to the nuclear genome and between organellar genomes occur extensively in flowering plants. The routes of the genetic materials flowing from one genome to another are biased, limited largely by compatibility of DNA replication and repair systems differing among the organelles and nucleus. After thoroughly surveying the tRNA gene transfer among organellar genomes and the nuclear genome of a domesticated rice (Oryza sativa L. ssp. indica), we found that (i) 15 mitochondrial tRNA genes originate from the plastid; (ii) 43 and 80 nuclear tRNA genes are mitochondrion-like and plastid-like, respectively; and (iii) 32 nuclear tRNA genes have both mitochondrial and plastid counterparts. Besides the native (or genuine) tRNA gene sets, the nuclear genome contains organelle-like tRNA genes that make up a complete set of tRNA species capable of transferring all amino acids. More than 97% of these organelle-like nuclear tRNA genes flank organelle-like sequences over 20 bp. Nearly 40% of them colocalize with two or more other organelle-like tRNA genes. Twelve of the 15 plastid-like mitochondrial tRNA genes possess 5′- and 3′-flanking sequences over 20 bp, and they are highly similar to their plastid counterparts. Phylogenetic analyses of the migrated tRNA genes and their original copies suggest that intergenomic tRNA gene transfer is an ongoing process with noticeable discriminatory routes among genomes in flowering plants. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Reviewing Editor: Dr. David Guttman     

Divergence of the mitochondrial leucyl tRNA synthetase genes in two closely related yeasts Saccharomyces cerevisiae and Saccharomyces douglasii: a paradigm of incipient evolution

Summary We studied the NAM2 genes of Saccharomyces douglasii and Saccharomyces cerevisiae, and showed that they are interchangeable for all the known functions of these genes, both mitochondrial protein synthesis and mitochondrial mRNA splicing. This confirms the prediction that the S. douglasii NAM2D gene encodes the mitochondrial leucyl tRNA synthetase (EC 6.1.1.4). The observation that these enzymes are interchangeable for their mRNA splicing functions, even though there are significant differences in the intron/exon structure of their mitochondrial genome, suggests that they may have a general role in yeast mitochondrial RNA splicing. A short open reading frame (ORF) precedes the synthetase-encoding ORF, and we showed that at least in S. cerevisiae this is not essential for the expression of the gene; however, it may be involved in a more subtle type of regulation. Sequence comparisons of S. douglasii and S. cerevisiae revealed a particularly interesting situation from the evolutionary point of view. It appears that the two yeasts have diverged relatively recently: there is remarkable nucleotide sequence conservation, with no deletions or insertions, but numerous (albeit non-saturating) silent substitutions resulting from transitions. This applies not only to the NAM2 coding regions, but also to two other ORFs flanking the NAM2 ORF. The regions between the ORFs (believed to be intergenic regions) are much less conserved, with several deletions and insertions. Thus S. douglasii and S. cerevisiae provide an ideal system for the study of molecular evolution, being two yeasts caught in the act of speciation.     

Expression of cytokinin biosynthetic isopentenyltransferase genes in Arabidopsis: tissue specificity and regulation by auxin, cytokinin, and nitrate

The rate-limiting step of cytokinin biosynthesis in Arabidopsis thaliana Heynh. is catalyzed by ATP/ADP isopentenyltransferases, A. thaliana IsoPentenyl Transferase (AtIPT)1, and AtIPT4, and by their homologs AtIPT3, AtIPT5, AtIPT6, AtIPT7, and AtIPT8. To understand the dynamics of cytokinins in plant development, we comprehensively analyzed the expression of isopentenyltransferase genes of Arabidopsis. Examination of their mRNA levels and the expression patterns of the beta-glucuronidase (GUS) gene fused to the regulatory sequence of each AtIPT gene revealed a specific expression pattern of each gene. The predominant expression patterns were as follows: AtIPT1::GUS, xylem precursor cell files in the root tip, leaf axils, ovules, and immature seeds; AtIPT3::GUS, phloem tissues; AtIPT4::GUS and AtIPT8::GUS, immature seeds with highest expression in the chalazal endosperm (CZE); AtIPT5::GUS, root primordia, columella root caps, upper part of young inflorescences, and fruit abscission zones; AtIPT7::GUS, endodermis of the root elongation zone, trichomes on young leaves, and some pollen tubes. AtIPT1, AtIPT3, AtIPT5, and AtIPT7 were downregulated by cytokinins within 4 h. AtIPT5 and AtIPT7 was upregulated by auxin within 4 h in roots. AtIPT3 was upregulated within 1 h after an application of nitrate to mineral-starved Arabidopsis plants. The upregulation by nitrate did not require de novo protein synthesis. We also examined the expression of two genes for tRNA isopentenyltransferases, AtIPT2 and AtIPT9, which can also be involved in cytokinin biosynthesis. They were expressed ubiquitously, with highest expression in proliferating tissues. These findings are discussed in relation to the role of cytokinins in plant development.     

Genetic control of translational fidelity in yeast: molecular cloning and analysis of the allosuppressor gene SAL3

Summary The fidelity of translation in the yeast Saccharomyces cerevisiae is controlled by a number of gene products. We have begun a molecular analysis of such genes and here describe the cloning and analysis of one of these genes, SAL3. Mutations at this locus, and at least four other unlinked loci (designated SAL1-SAL5), increase the efficiency of the tRNA ochre suppressor SUQ5, and are thus termed allosuppressors. We have cloned the SAL3 gene from a yeast genomic library by complementation of a sal3 mutation. Integration of the cloned sequence into the yeast chromosome was used to confirm that the SAL3 gene had been cloned. SAL3 gene is present in a single copy in the yeast genome, is transcribed into a 2.3-kb polyadenylated mRNA and encodes a protein of M_r 80 000. The size of the SAL3 gene product strongly suggests that it is not a ribosomal protein.     

Restoration of shooty morphology of a nontumorous mutant of Nicotiana glauca x N. langsdorffii by cytokinin and the isopentenyltransferase gene

The shooty morphology of a nontumorous amphidiploid mutant of Nicotiana glauca Grah. x N. langsdorffii Weinm. was restored by cytokinins, whether exogenously applied or endogenously produced by transformation of the mutant with a transfer DNA (T-DNA) cytokinin-biosynthesis gene (isopentenyltransferase; ipt). Auxins alone did not confer this effect. Similar transformation was not achieved for the parental species. In the case of transformation with the ipt gene, selection of the transformed tissues was based on its hormone-independent growth in the presence of the antibiotic kanamycin. Transformed tissues exhibited a shooty morphology, indistinguishable from that of wildtype genetic tumors N. glauca x N. langsdorffii. This altered phenotype was caused by the presence and constitutive expression of the ipt gene. The insertion and expression of this gene in transformed tissues was confirmed by using the polymerase chain reaction (PCR) technique as well as conventional molecular hybridization analysis. Expression of the ipt gene led to an elevated level of cytokinin in the transformed mutant tissues. This evidence supports the notion that genetic tumors are caused, at least in part, by elevated levels of cytokinin in interspecific hybrids.     

Mutations in the NAM2 genes of Saccharomyces cerevisiae and S. douglasii are clustered non-randomly as a result of constraints on the nucleic acid sequence and not on the protein

Summary We present a statistical study of the nature and distribution of mutations along the NAM2 gene coding for the mitochondrial leucyl tRNA synthetase in Saccharomyces cerevisiae and S. douglasii (Herbert et al. 1988). Two important facts are observed: (1) the relative frequency of transitions and transversions is the same among silent substitutions and replacements. (2) The two kinds of mutations (silent substitutions and replacements) are distributed in the same way along the gene. This distribution is not random; the mutations are clustered and the clusters are regularly spaced along the gene. The NAM2 gene offers an example spaced along the gene. The NAM2 gene offers an example of recent divergence. We show that, in this case, the fixation of mutations is the result of genetic drift and of constraints on the nucleic acid sequence and not on that of the protein.     

Identification of two additional members of the tRNA isopentenyltransferase family in Physcomitrella patens

The Physcomitrella patens genome has seven genes apparently coding for the isopentenyltransferase type of tRNA-modifying enzyme, while other organisms have one or two. The predicted sequences have parts that differ significantly from other isopentenyltransferases. Only one of the seven (PpIPT1) has earlier been shown to be expressed. We now report expression of two more, PpIPT4 and PpIPT5. The cloned genes were able to functionally complement a yeast mutant lacking tRNA isopentenyltransferase. Sequencing showed they are related to the earlier studied PpIPT1. The sequences of the three differ mainly from each other in a tRNA-binding area and the 5′-end subcellular targeting motif area. This indicates that, after arising through gene duplication, they have evolved to enable partly different functions.     

A new method for enzymatic preparation of isopentenyladenine-type and<Emphasis Type="Italic"> trans</Emphasis>-zeatin-type cytokinins with radioisotope-labeling

We describe a new enzymatic reaction method for the preparation of the radioisotope-labeled cytokinins isopentenyladenine (iP), trans-zeatin (tZ), and their ribosides. The method is based on the three enzyme activities of an adenylate isopentenyltransferase (IPT; EC 2.5.1.27) from Arabidopsis thaliana, an alkaline phosphatase (EC 3.1.3.1) from calf intestine, and a purine-nucleoside phosphorylase (EC 2.4.2.1) from Escherichia coli. The A. thaliana IPT, AtIPT7, utilized both dimethylallyldiphosphate and 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate as isoprenoid donors. The dual specificity of the substrates enabled us to produce iP-type and tZ-type cytokinins separately in the same system simply by switching the substrates. Our method affords a much higher yield of the labeled products than the chemical reaction methods previously used. These labeled compounds will be useful tools for cytokinin research, such as receptor–ligand assays and cell metabolism studies.     

Transfer RNA Is the Source of Extracellular Isopentenyladenine in a Ti-Plasmidless Strain of Agrobacterium tumefaciens

Even in the absence of the classical Ti plasmid-encoded cytokinin biosynthetic genes ipt and tzs, Agrobacterium tumefaciens strains still release significant amounts of the cytokinin isopentenyladenine (iP) into the culture medium (R.W. Kaiss-Chapman and R.O. Morris [1977] Biochem Biophys Res Commun 76: 453-459). A potential source of the iP is isopentenylated transfer RNA (tRNA), which, in turn, is synthesized by the activity of tRNA:isopentenyltransferase encoded by the bacterial miaA gene. To determine whether secreted iP had its origin in isopentenylated tRNA, a miaA- deletion/insertion mutant was prepared and reconstructed in Agrobacterium tumefaciens in vivo. The mutant no longer possessed tRNA:isopentenylation activity and no longer released iP into the extracellular medium. Transfer RNA therefore makes a small but significant contribution to the total amount of cytokinin normally secreted by Agrobacterium strains. tRNA-mediated synthesis may also account for cytokinin production by other plant-associated bacteria, such as Rhizobia, that have been reported to secrete similarly low levels of nonhydroxylated cytokinins.     

The gene fimU affects expression of Salmonella typhimurium type 1 fimbriae and is related to the Escherichia coli tRNA gene argU

The gene fimU, located on a recombinant plasmid carrying the Salmonella typhimurium type 1 fimbrial gene cluster is closely related to the Escherichia coli tRNA gene argU. The fimU gene complements an E. coli argU mutant that is a P2 lysogen, thereby allowing the phage P4 to grow in this strain but preventing the growth of phage lambda. In addition, fimU was shown to be involved in fimbrial expression since transformants of the E. coli argU mutant could produce fimbriae only in the presence of fimU but not in its absence, whereas in an E. coli argU ⁺ strain fimbriation did not require the fimU gene.     

Plant mitochondrial RNase P

Molecular investigations in mitochondria of higher plants have to take in account the complicated genomic structure of these organelles and their complex mode of gene expression. Recently tRNA processing activities and particulary RNase P-like activities have been described for mitochondria of mono- and dicot plants. The determined biochemical characteristics of these plant mitochondrial tRNA processing enzymes now allow a comparison to the bacterial prototype from which they evolved. The substrate specifity of the plant mitochondrial RNase P in particular has unique selection parameters distinct from theE. coli RNase P.