Tristan A. Dyer

Delayed Leaf Senescence in Tobacco Plants Transformed with tmr, a Gene for Cytokinin Production in Agrobacterium

The aim of this study was to investigate whether enhanced levels of endogenous cytokinins could influence plant development, particularly leaf senescence. Tobacco plants were transformed with the Agrobacterium tumefaciens gene tmr, under the control of the soybean heat shock promoter HS6871. This gene encodes the enzyme isopentenyl transferase, which catalyzes the initial step in cytokinin biosynthesis. After heat shock, the cytokinin level increased greatly and the level of tmr mRNA, undetectable at 20[deg]C, rose and remained high for up to 8 hours. The levels of cytokinin and tmr mRNA were substantially lower by 24 hours. Transformed plants grown at 20[deg]C were shorter, had larger side shoots, and remained green for longer than untransformed plants. The differences were more pronounced after several heat shocks of whole plants or defined areas of leaves. Our results demonstrated that plant morphology and leaf senescence can be manipulated by changing the endogenous level of cytokinins.

Use of the polymerase chain reaction to detect spacer size heterogeneity in plant 5S-rRNA gene clusters and to locate such clusters in wheat (Triticum aestivum L.)

SummaryWe have used the polymerase chain reaction to analyse variation in the size of individual 5S-ribosomal gene spacer sequences. This reaction can be used to demonstrate inter- and intraspecific variation in spacer size, and combined with DNA sequencing it may thus be a valuable taxonomic tool. Two sets of nested polymerase chain reaction primers were designed to amplify the nontranscribed spacer DNA between repeated 5S-rRNA genes. These “universal” primers were used to generate fragments from the genomic DNA from several unrelated monocotyledonous plants. Ribosomal RNA spacer sequences generated in these experiments could also be used to locate 5S-rRNA gene clusters on specific chromosomes in hexaploid wheat (Triticum aestivum). Three distinct spacer sizes were observed after amplification. These were assigned locations on chromosomes by analysing amplification products of genomic DNA from nullisomic/tetrasomic and ditelosomic wheat stocks. “Large” 508-bp 5S repeats are located on the short arm of chromosome 5B and “short” 416-bp and 425-bp repeat unit variants are located on the short arms of chromosomes 1B and 1D, respectively. No other loci were detected. The spacer fragments were cloned, sequenced, and shown to be homologous to wheat 5S-rRNA spacers previously identified. Spacers of uniform size but with some sequence heterogeneity were shown to be located at each locus.

A physical map of wheat chloroplast DNA showing the location of the structural genes for the ribosomal RNAs and the large subunit of ribulose 1,5-bisphosphate carboxylase

SummaryThe restriction endonucleases SalGI and PstI have been used to construct a physical map of wheat ctDNA. The molecule was found to contain approximately 135 kbq, and in common with many other higher plant ctDNAs about 15% of the sequences are repeated in an inverted orientation. It was established by electron microscopy that, in wheat, each segment of the inverted repeat contains 21.0 kbp, and that the single copy regions separating the two repeated segments contain 12.8 kbp and 80.2 kbp. Blot hybridisation showed that one set of ribosomal genes is located in each segment of the inverted repeat region and the sizes of these genes were accurately determined by measuring the dimensions of hybrids between the chloroplast rRNAs and the identified Sal and Eco fragments on electron micrographs: the genes for the 16S and 23S rRNAs contain 1530 bp and 2850 bp respectively and are separated by a spacer region of 2350 bp. The Bgl fragment of maize ctDNA known to contain the structural gene for the large-subunit (LS) of ribulose 1,5-bisphosphate carboxylase was used as a probe to locate the LS gene in wheat ctDNA. A small (2.8 kbp) Eco fragment was found to contain most of the wheat LS gene and is derived from the larger single-copy region, 23.5 kbp away from one segment of the inverted repeat and 54.8 kbp from the other.

Common features of three inversions in wheat chloroplast DNA

SummaryWe have determined the DNA sequences of regions involved in two of the three inversions known to have occurred during the evolution of wheat chloroplast DNA. This establishes the extent of the second largest of the three inversions. Examination of these sequences suggests that although short repeated sequences are present, the endpoints of the second and third inversions are not associated with repeated sequences as long as those associated with the first inversion. However the endpoints of all three inversions are all adjacent to at least one tRNA gene, and there is evidence that three of the tRNA genes have been subjected to partial duplication, possibly at the time of inversion. This suggests that tRNA genes might be involved with rearrangements of chloroplast DNA, as has also been postulated for mitochondrial DNA.

DRL1, a Homolog of the Yeast TOT4/KTI12 Protein, Has a Function in Meristem Activity and Organ Growth in Plants

The DEFORMED ROOTS AND LEAVES1 (DRL1) gene is single copy in the Arabidopsis genome, and based on overall amino acid similarity and conservation of functional domains, the DRL1 protein is homologous with yeast TOT4/KTI12. TOT4/KTI12 associates with Elongator, a multisubunit complex that binds the RNA polymerase II transcription elongation complex. Recessive mutations at the DRL1 locus caused defective organ formation indicative of disorganized shoot, inflorescence, flower, and root meristems. DRL1 is a putative ATP/GTP binding protein; in addition, calmodulin binding activity was demonstrated in vitro for the C terminus of the DRL1 protein. Phenotypic and genetic data position DRL1 relative to regulatory loci for leaf development, in which it acts early. We identified Arabidopsis homologs for the six Elongator components and hypothesize that DRL1 regulates transcription elongation through a putative plant Elongator. Upregulation of the ANGUSTIFOLIA transcript in the strong drl1-2 allele supports this model.

Location and nucleotide sequence of the gene for cytochrome f in wheat chloroplast DNA

SummaryThe gene for cytochrome f has been located in wheat chloroplast DNA by hybridisation with a 3.3 kbp BglII fragment of pea chloroplast DNA containing the gene for cytochrome f, by in vitro transcription-translation of cloned restriction fragments of wheat chloroplast DNA and by nucleotide sequence analysis. The gene is located 3 kbp from the 3′ end of the gene for the large subunit of ribulose bisphosphate carboxylase and is transcribed from the same DNA strand. Nucleotide sequence analysis reveals an open reading frame of 320 amino acids, of which 285 amino acid residues comprise the mature polypeptide and 35 amino acid residues probably represent an N-terminal signal sequence. The nucleotide sequence of the wheat gene shows 85% homology with the gene for pea cytochrome f.

Nucleotide sequences of the genes for the alpha, beta and epsilon subunits of wheat chloroplast ATP synthase

SummaryThe nucleotide sequences of the chloroplast genes for the alpha, beta and epsilon subunits of wheat chloroplast ATP synthase have been determined. Open reading frames of 1512 bp, 1494 bp and 411 bp are deduced to code for polypeptides of molecular weights 55201, 53796 and 15200, identified as the alpha, beta and epsilon subunits respectively by homology with the subunits from other sources and by amino acid sequencing of the epsilon subunit. The genes for the beta and epsilon subunits overlap by 4 bp. The gene for methionine tRNA is located 118 bp downstream from the epsilon subunit gene. Comparisons of the deduced amino acid sequences of the alpha and beta subunits with those from other species suggest regions of the proteins involved in adenine nucleotide binding.

Localization of wheat chloroplast genes for the beta and epsilon subunits of ATP synthase

SummaryCloned restriction fragments from wheat chloroplast DNA have been shown to direct the synthesis, in a cell-free E. coli transcription-translation system, of the beta and epsilon subunits of the CF1 component of ATP synthase. The products were identified by immunoprecipitation, their mobility in SDS-polyacrylamide gels and peptide mapping of limited proteolytic digests. Genes for these subunits have been located in the large single-copy region of wheat chloroplast DNA, very close to the gene for the large subunit of ribulose bisphosphate carboxylase.

Cloning and Expression Analysis of the Plastidic Fructose-1,6-Bisphosphatase Coding Sequence from Potato - Circumstantial Evidence for the Import of Hexoses into Chloroplasts

A copy DNA encoding the plastid-located isoform of the fructose-1,6-bisphosphatase (cp-FBPase) has been cloned from potato (Solanum tuberosum L.). Sequence analysis reveals a high degree of homology to cp-FBPases from wheat, spinach, andArabidopsis. Analysis of RNA blots shows that the expression of the cp-FBPase is limited to green tissue such as leaf and stem, and is absent from photosynthetically inactive tissue such as roots, tubers and stolons. This provides additional evidence that hexoses or hexose phosphates are imported into amyloplasts of heterotrophic tissues. Incubation of detached leaves of potato in darkness in a sucrosecontaining medium leads to massive accumulation of both starch and transcripts encoding starch biosynthetic enzymes. However, no transcripts encoding the cp-FBPase are detectable under these conditions.

DEVELOPMENTAL, CIRCADIAN AND LIGHT REGULATION OF WHEAT FERREDOXIN GENE EXPRESSION

A genomic clone encoding the precursor of wheat leaf ferredoxin has been isolated and characterised. The uninterrupted PetF gene encodes a polypeptide of 143 amino acid residues, consisting of an N-terminal presequence of 46 amino acid residues and a mature polypeptide of 97 amino acid residues. Southern blot analysis suggests that six copies of the PetF gene are present in the wheat haploid genome. Northern blot analysis has shown that the genes are both developmentally and light regulated in wheat seedlings and provides evidence that a circadian rhythm regulates the steady-state levels of ferredoxin transcripts. The intact wheat gene and several chimeric constructs, containing portions of the 5′-upstream region fused to the β-glucuronidase reporter gene, have been introduced into tobacco plants, but levels of β-glucuronidase activity above background were not detected, suggesting that the 5′-upstream region is unable to function as a promoter in tobacco plants.