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D J Llewellyn

Publications and source records attributed to D J Llewellyn.

At least 19 recordsLinked to original sources
PubMed2001

The control of single-celled cotton fiber elongation by developmentally reversible gating of plasmodesmata and coordinated expression of sucrose and K+ transporters and expansin.

Each cotton fiber is a single cell that elongates to 2.5 to 3.0 cm from the seed coat epidermis within approximately 16 days after anthesis (DAA). To elucidate the mechanisms controlling this rapid elongation, we studied the gating of fiber plasmodesmata and the expression of the cell wall-loosening gene expansin and plasma membrane transporters for sucrose and K(+), the major osmotic solutes imported into fibers. Confocal imaging of the membrane-impermeant fluorescent solute carboxyfluorescein (CF) revealed that the fiber plasmodesmata were initially permeable to CF (0 to 9 DAA), but closed at approximately 10 DAA and re-opened at 16 DAA. A developmental switch from simple to branched plasmodesmata was also observed in fibers at 10 DAA. Coincident with the transient closure of the plasmodesmata, the sucrose and K(+) transporter genes were expressed maximally in fibers at 10 DAA with sucrose transporter proteins predominately localized at the fiber base. Consequently, fiber osmotic and turgor potentials were elevated, driving the rapid phase of elongation. The level of expansin mRNA, however, was high at the early phase of elongation (6 to 8 DAA) and decreased rapidly afterwards. The fiber turgor was similar to the underlying seed coat cells at 6 to 10 DAA and after 16 DAA. These results suggest that fiber elongation is initially achieved largely by cell wall loosening and finally terminated by increased wall rigidity and loss of higher turgor. To our knowledge, this study provides an unprecedented demonstration that the gating of plasmodesmata in a given cell is developmentally reversible and is coordinated with the expression of solute transporters and the cell wall-loosening gene. This integration of plasmodesmatal gating and gene expression appears to control fiber cell elongation.

Base Sequence↗
PubMed1999

A TM3-like MADS-box gene from Eucalyptus expressed in both vegetative and reproductive tissues.

MADS-box genes in plants are a diverse class of transcription factors that are involved in regulating developmental processes, particularly meristem and organ identity during floral development. They are characterized by a highly conserved MADS-box domain of 59 amino acids that binds to specific DNA sequences. We report the characterization of a cDNA clone, ETL (Eucalyptus TM3 Like), from Eucalyptus globulus subspecies bicostata encoding a putative transcription factor of the MADS-box class that is strongly expressed in both vegetative and floral tissues, suggesting that it regulates processes other than floral development. The clone was isolated from a floral bud cDNA library with a probe generated from Eucalyptus genomic DNA by PCR using degenerate primers to the MADS-box of the floral regulatory gene APETALA 1. The ETL cDNA clone encodes a putative protein of 206 amino acids that contains an N-terminal MADS-box and a helical domain of approx. 60 amino acids predicted to form a coiled-coil (K-box). These structural features are characteristic of plant MADS-box proteins. The MADS-box domain contains all the signature residues of a class of MADS-box genes typified by the tomato gene TM3 and overall, ETL shows 56% amino acid identity to TM3. Like TM3, the ETL gene is expressed in both vegetative and reproductive organs, predominantly in root and shoot meristems and organ primordia, as well as in developing male and female floral organs.

Amino Acid Sequence↗
PubMed1998

Eucalyptus has a functional equivalent of the Arabidopsis floral meristem identity gene LEAFY.

Two genes cloned from Eucalyptus globulus, Eucalyptus LeaFy (ELF1 and ELF2), have sequence homology to the floral meristem identity genes LEAFY from Arabidopsis and FLORICAULA from Antirrhinum. ELF1 is expressed in the developing eucalypt floral organs in a pattern similar to LEAFY while ELF2 appears to be a pseudo gene. ELF1 is expressed strongly in the early floral primordium and then successively in the primordia of sepals, petals, stamens and carpels. It is also expressed in the leaf primordia and young leaves and adult and juvenile trees. The ELF1 promoter coupled to a GUS reporter gene directs expression in transgenic Arabidopsis in a temporal and tissue-specific pattern similar to an equivalent Arabidopsis LEAFY promoter construct. Strong expression is seen in young flower buds and then later in sepals and petals. No expression was seen in rosette leaves or roots of flowering plants or in any non-flowering plants grown under long days. Furthermore, ectopic expression of the ELF1 gene in transgenic Arabidopsis causes the premature conversion of shoots into flowers, as does an equivalent 35S-LFY construct. These data suggest that ELF1 plays a similar role to LFY in flower development and that the basic mechanisms involved in flower initiation and development in Eucalyptus are similar to those in Arabidopsis.

Amino Acid Sequence↗
PubMed1997

The Arabidopsis thaliana genome has multiple divergent forms of phosphoinositol-specific phospholipase C1.

Highly degenerate primers to conserved regions of the eukaryotic phosphoinositol-specific phospholipase C (PLC) were used to amplify fragments of plant PLCs from Arabidopsis thaliana genomic DNA. Eight completely different fragment sequences that showed high homology to PLCs of both animals and plants were isolated. The variation between these putative PLCs was high and suggests that, like animals, plants have multiple isoforms of PLC. Using one of the PCR clones, we isolated a corresponding full-length Arabidopsis PLC gene (ATHATPLC1G), and sequence analysis indicated that it was most like a delta-type PLC. This gene is 2.5 kb and contains seven introns, all but one of which has intron/exon border sequences that conform to the Arabidopsis consensus. The structural complexity of the gene is relatively simple compared to mammalian beta-type PLCs that can be 15 kb long with up to 30 introns. The plant gene is a single copy and was mapped to four Arabidopsis YACs, one located on chromosome 2. The promoter region contained two TATA-like elements at -43 and -185 and other putative regulatory elements that suggest that this PLC is hormonally regulated. This is the first plant PLC gene and the first delta type-PLC gene from a higher organism to be sequenced.

Amino Acid Sequence↗
PubMed1997

Two hemoglobin genes in Arabidopsis thaliana: the evolutionary origins of leghemoglobins.

We cloned two hemoglobin genes from Arabidopsis thaliana. One gene, AHB1, is related in sequence to the family of nonsymbiotic hemoglobin genes previously identified in a number of plant species (class 1). The second hemoglobin gene, AHB2, represents a class of nonsymbiotic hemoglobin (class 2) related in sequence to the symbiotic hemoglobin genes of legumes and Casuarina. The properties of these two hemoglobins suggest that the two families of nonsymbiotic hemoglobins may differ in function from each other and from the symbiotic hemoglobins. AHB1 is induced, in both roots and rosette leaves, by low oxygen levels. Recombinant AHB1 has an oxygen affinity so high as to make it unlikely to function as an oxygen transporter. AHB2 is expressed at a low level in rosette leaves and is low temperature-inducible. AHB2 protein has a lower affinity for oxygen than AHB1 but is similar to AHB1 in having an unusually low, pH-sensitive oxygen off-rate.

Amino Acid Sequence↗
PubMed1997

Isolation of the glucose oxidase gene from Talaromyces flavus and characterisation of its role in the biocontrol of Verticillium dahliae.

The glucose oxidase gene from the biocontrol fungus Talaromyces flavus has been isolated and shown to be only 64% identical at the amino-acid sequence level to the similar enzyme from Aspergillus niger. A transformation system has been developed for both T. flavus and the related T. macrosporus and has been used to create Talaromyces spp. which either over-express or are deficient in glucose oxidase. In vitro inhibition experiments on Verticillium dahliae using culture filtrates from these transformants indicates that secreted glucose oxidase is responsible for a large part of the growth inhibition of V. dahliae microsclerotia and hyphae by T. flavus, although other inhibitory compounds may also play a role. In pot trials with cotton plants, both Talaromyces species had some biocontrol activity, but there was no significant difference in the incidence of Verticillium wilt with either the presence or absence of glucose oxidase activity in the biocontrol fungus. Under the experimental conditions used, insufficient glucose is presumably present in the soil around cotton roots to generate sufficient hydrogen peroxide to inhibit V. dahliae and the observed biocontrol activity must be attributed to other factors.

Amino Acid Sequence↗
PubMed1997

Cell-specific expression of the promoters of two nonlegume hemoglobin genes in a transgenic legume, Lotus corniculatus.

The promoters of the hemoglobin genes from the nitrogen-fixing tree Parasponia andersonii and the related nonnitrogen-fixing Trema tomentosa both confer beta-glucuronidase reporter gene expression to the central zone of the nodules of a transgenic legume, Lotus corniculatus. beta-Glucuronidase expression was high in the uninfected interstitial cells and parenchyma of the surrounding boundary layer and was low in the Rhizobium-infected cells. This contrasts with the expression of both the P. andersonii hemoglobin protein in P. andersonii nodules and the endogenous Lotus leghemoglobins that are expressed in the infected cells at very high levels. The expression pattern of the P. andersonii and T. tomentosa hemoglobin promoters in L. corniculatus resembles that of a nonsymbiotic hemoglobin gene from Casuarina glauca, which was introduced into this legume, and suggests that only the nonsymbiotic functions of the P. andersonii promoter are being recognized. Deletion of the distal segments of both the P. andersonii and T. tomentosa promoters identified regions important for the control of their tissue-specific and temporal activity in Lotus. Potential regulatory elements, which enhance nodule expression and suppress nonnodule expression, were also identified and localized to a distal promoter segment. A proximal AAGAG motif is present in the P. andersonii, T. tomentosa, and nonsymbiotic Casuarina hemoglobin genes. Mutation of this motif in the P. andersonii promoter resulted in a significant reduction in both the nodule and root expression levels in L. corniculatus. Some of the regulatory motifs characterized are similar to, but different from, the nodulin motifs of the leghemoglobins.

Base Sequence↗
PubMed1996

Isolation and characterization of full-length cDNA clones of the giant taro (Alocasia macrorrhiza) trypsin/chymotrypsin inhibitor.

A full-length cDNA encoding the 206 amino acid open reading frame of a trypsin/chymotrypsin inhibitor abundant in the corms of giant taro (Alocasia macrorrhiza) was isolated. An internal fragment was cloned using degenerate primers corresponding to a region of the mature protein sequence and the 'rapid amplification of cDNA ends' (RACE) method used to generate a composite cDNA sequence. The length of the cDNA was close to the predicted size of the corresponding transcript deduced from northern blot analysis of corm mRNA. The inhibitor was expressed strongly in the mature corm, at low levels in leaf blades and petioles but not in roots. Southern blot analysis of the giant taro DNA indicated that this inhibitor is encoded by a small multigene family and this was further supported by the isolation of two different sequence classes from corm cDNA using primers to the ends of the composite sequence.

Amino Acid Sequence↗
PubMed1993

Does the ocs-element occur as a functional component of the promoters of plant genes?

The structural requirements of the ocs-element, a promoter element in several genes transferred to the host plant nucleus by Agrobacterium tumefaciens and certain DNA viruses, have been further characterized both in vitro and in vivo. Two adjacent and functionally identical protein-binding sites separated by an exact number of nucleotides are required for in vivo activity of the ocs-element. Plant pathogens have presumably recruited cellular transcription factors that interact with these binding sites to drive the high-level expression of their essential genes. Our functional analyses of the ocs-elements from two pathogen promoters define the structure of a sequence motif that might also be expected to occur in plant nuclear genes, and a search of the plant gene database has identified a number of plant gene promoters that contain sequences that resemble the ocs-element. These sequences were analysed for their ability both to bind the maize nuclear protein OCSTF and to activate transcription of an inactive promoter. A functional ocs-element was identified in only one of the plant genes, the soybean heat-shock gene, Gmhsp26-A. The apparent rarity of the ocs-element in plant genes contrasts with its frequent use by pathogens that transform the plant nucleus. Sequences resembling half of an ocs-element, on the other hand, are common in plant promoters and may form part of multi-element control motifs with a variety of regulatory functions. Plant pathogens may, therefore, have evolved to circumvent tight regulatory control of their promoters by the host by duplicating the half ocs-element promoter motifs to take advantage of the ubiquitous ocs-element-binding transcription factors in plants.

Agrobacterium tumefaciens↗
PubMed1993

Isolation of a maize bZIP protein subfamily: candidates for the ocs-element transcription factor.

Ocs-elements, a family of 20 bp DNA sequences, are components of a number of promoters active in plants. In the maize BMS cell line the dominant ocs-element binding activity is the ocs-element transcription factor complex called OTF. The isolation of cDNA clones from a BMS cDNA expression library for two bZIP (basic region-leucine zipper) proteins that bind the ocs-element sequence and are good candidates for forming at least part of OTF is described. The two ocs-element binding proteins, called OBF3.1 and OBF3.2, are closely related, with the OBF3.1 protein sharing 95.8% amino acid homology with part of the OBF3.2 protein although there were significant differences in the 3' untranslated regions. Genomic Southern blot analysis revealed a small gene family with a minimum of two OBF3 loci mapping to chromosomes 3L105 and 8L075. The OBF3.1 protein shared considerable homology with the wheat HBP1b protein (80% amino acid identity) and to a lesser extent with the tobacco TGA1aa protein. OBF3.1 like HBP1b was able to bind well to the Hex sequence but poorly to G-box/ABRE sequences. Interestingly, OBF3.1 bound eightfold more efficiently to an ocs-element sequence than TGA1a, raising the possibility that OBF3.1 and TGA1a may be distinct members of an OBF3/TGA subfamily.

Amino Acid Sequence↗
PubMed1990

Nonlegume hemoglobin genes retain organ-specific expression in heterologous transgenic plants.

Hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa have been isolated [Landsmann et al. (1986). Nature 324, 166-168; Bogusz et al. (1988). Nature 331, 178-180]. The promoters of these genes have been linked to a beta-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus. Both promoters directed root-specific expression in transgenic tobacco. When transgenic Lotus plants were nodulated by Rhizobium loti, both promoter constructs showed a high level of nodule-specific expression confined to the central bacteroid-containing portion of the nodule corresponding to the expression seen for the endogenous Lotus leghemoglobin gene. The T. tomentosa promoter was also expressed at a low level in the vascular tissue of the Lotus roots. The hemoglobin promoters from both nonlegumes, including the non-nodulating species, must contain conserved cis-acting DNA signals that are responsible for nodule-specific expression in legumes. We have identified sequence motifs postulated previously as the nodule-specific regulatory elements of the soybean leghemoglobin genes [Stougaard et al. (1987). EMBO J. 6, 3565-3569].

Base Sequence↗
PubMed1990

OCSBF-1, a maize ocs enhancer binding factor: isolation and expression during development.

The ocs-elements comprise a family of related 20-base pair DNA sequences with dyad symmetry that are functional components of the promoters of several genes introduced into the plant nucleus by Agrobacterium transformation or infection by DNA viruses. We describe the isolation and characterization of a maize cDNA that encodes a protein, OCSBF-1, that binds specifically to ocs-element sequences. The 21-kilodalton OCSBF-1 protein was encoded by a single copy, intron-less gene. The gene was differentially expressed in maize plants. Developing leaves had a gradient of OCSBF-1 mRNA with the basal portion of the leaves, which contain dividing and differentiating cells, having 40-fold to 50-fold higher levels of OCSBF-1 transcripts than the apical portion of the leaves, where the cells are fully differentiated. Roots and shoots of young plants had levels of OCSBF-1 mRNA similar to the basal portions of developing leaves. OCSBF-1 contained a small basic amino acid region and a potential leucine zipper motif homologous to the DNA-binding domains of the basic region-leucine zipper family of transcription factors such as Jun and GCN4. A truncated protein with the amino-terminal 76 amino acids of OCSBF-1, encompassing the basic domain and leucine zipper motif, still bound to ocs-element sequences in vitro. OCSBF-1 was able to bind to a site within each half of the ocs-element as well as to animal AP-1 and CREB sites.

Amino Acid Sequence↗
PubMed1989

The ocs-element is a component of the promoters of several T-DNA and plant viral genes.

The ocs-element is an enhancer element first identified in the promoter of the octopine synthase gene (OCS) where it occurs as a 16 bp palindromic sequence. The transcriptional enhancing activity of the ocs-element correlated with in vitro binding of a transcription factor. We have now identified ocs-elements in the promoter regions of six other T-DNA genes involved in opine synthesis and three plant viral promoters including the 35S promoter of cauliflower mosaic virus. These elements bind the ocs transcription factor in vitro and enhance transcription in plant cells. Comparison of the sequences of these 10 elements has defined a 20 bp consensus sequence, TGACG(T/C)AAG(C/G)(G/A)(A/C)T(G/T)ACG(T/C)(A/C)(A/C), which includes the 16 bp palindrome in its central region. We propose the name ocs-element for this class of promoter elements of similar sequence and function.

Base Sequence↗
PubMed1989

Expression of a bacterial gene in transgenic tobacco plants confers resistance to the herbicide 2,4-dichlorophenoxyacetic acid.

Plants resistant to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were produced through the genetic engineering of a novel detoxification pathway into the cells of a species normally sensitive to 2,4-D. We cloned the gene for 2,4-D monooxygenase, the first enzyme in the plasmid-encoded 2,4-D degradative pathway of the bacterium Alcaligenes eutrophus, into a cauliflower mosaic virus 35S promoter expression vector and introduced it into tobacco plants by Agrobacterium-mediated transformation. Transgenic tobacco plants expressing the highest levels of the monooxygenase enzyme exhibited increased tolerance to 2,4-D in leaf disc and seed germination assays, and young plants survived spraying with levels of herbicide up to eight times the usual field application rate. The introduction of the gene for 2,4-D monooxygenase into broad-leaved crop plants, such as cotton, should eventually allow 2,4-D to be used as an inexpensive post-emergence herbicide on economically important dicot crops.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1987

Structure and expression of an alcohol dehydrogenase 1 gene from Pisum sativum (cv. "Greenfeast").

Three genomic clones for anaerobically inducible alcohol dehydrogenase (Adh) have been isolated from Pisum sativum cv. "Greenfeast" via cDNA cloning. One of these contains a complete gene, has exon sequences corresponding to one of the cDNA sequences and is likely to be an expressed gene. This gene has a structure similar to the Adh genes of maize, with introns in the same positions in the coding sequence but differing in their lengths and nucleotide sequences. At the nucleotide level the coding sequence is 75% homologous to both maize Adh1 and Adh2 and 80% homologous to the Adh gene from Arabidopsis, but has an extra coding triplet in exon 1 that is not found in the other plant Adh genes. The non-translated regions of all the gene transcripts are widely divergent between species. A short segment of the pea Adh promoter region (-290 to +57) was fused to a reporter gene and introduced into protoplasts of Nicotiana plumbaginifolia by electroporation. Transient expression of the introduced gene increased markedly when the transfected protoplasts were incubated under anaerobic conditions, showing that cis-acting regulatory signals necessary for anaerobic control of expression reside in the -290 to +57 segment. Sequence comparisons between this region and the corresponding regions of maize and Arabidopsis Adh genes have identified short sequences that may be involved in the anaerobic regulation of plant Adh genes.

Alcohol Oxidoreductases↗
PubMed1987

The ocs element: a 16 base pair palindrome essential for activity of the octopine synthase enhancer.

A 176 bp DNA sequence lying upstream of the octopine synthase (ocs) promoter, previously shown to have enhancer-like properties in transgenic tobacco [Ellis et al. (1987) EMBO J., 6, 11-16], functions as an enhancer in protoplasts of Zea mays (a monocot plant) and Nicotiana plumbaginifolia (a dicotplant). We have characterized this element by transient expression assays using a linked alcohol dehydrogenase (Adh1) promoter from Z. mays and the chloramphenicol acetyltransferase coding sequences. The ocs sequence functions in both orientations but its enhancing activity is dependent upon its distance from the Adh1 promoter. Transient expression assays using deletion mutants and synthetic oligonucleotides show that a 16 bp palindrome ACGTAAGCGCTTACGT, contained within the 176 bp fragment, is essential and sufficient for enhancing activity in transient expression assays.

Journal Article↗
PubMed1987

Maize Adh-1 promoter sequences control anaerobic regulation: addition of upstream promoter elements from constitutive genes is necessary for expression in tobacco.

The promoter region of a maize alcohol dehydrogenase gene (Adh-1) was linked to a reporter gene encoding chloramphenicol acetyl transferase (CAT) and transformed stably into tobacco cells using T-DNA vectors. No CAT enzyme activity could be detected in transgenic tobacco plants unless upstream promoter elements from the octopine synthase gene or the cauliflower mosaic virus 35S promoter were supplied in addition to the maize promoter region. CAT enzyme activity and transcription of the chimaeric gene were then readily detected after anaerobic induction. The first 247 bp upstream of the translation initiation codon of the maize Adh-1 gene were sufficient to impose anaerobic regulation on the hybrid gene and S1 nuclease mapping confirmed mRNA initiation is from the normal maize Adh-1 transcription start point.

Alcohol Dehydrogenase↗