Biomedical subjects

G Pilate

Publications and source records attributed to G Pilate.

11 recordsLinked to original sources

PubMed2006

The genome of black cottonwood, Populus trichocarpa (Torr. & Gray).

We report the draft genome of the black cottonwood tree, Populus trichocarpa. Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. More than 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication event; about 8000 pairs of duplicated genes from that event survived in the Populus genome. A second, older duplication event is indistinguishably coincident with the divergence of the Populus and Arabidopsis lineages. Nucleotide substitution, tandem gene duplication, and gross chromosomal rearrangement appear to proceed substantially more slowly in Populus than in Arabidopsis. Populus has more protein-coding genes than Arabidopsis, ranging on average from 1.4 to 1.6 putative Populus homologs for each Arabidopsis gene. However, the relative frequency of protein domains in the two genomes is similar. Overrepresented exceptions in Populus include genes associated with lignocellulosic wall biosynthesis, meristem development, disease resistance, and metabolite transport.

Arabidopsis↗

PubMed2001

Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR.

Studying lignin-biosynthetic-pathway mutants and transgenics provides insights into plant responses to perturbations of the lignification system, and enhances our understanding of normal lignification. When enzymes late in the pathway are downregulated, significant changes in the composition and structure of lignin may result. NMR spectroscopy provides powerful diagnostic tools for elucidating structures in the difficult lignin polymer, hinting at the chemical and biochemical changes that have occurred. COMT (caffeic acid O-methyl transferase) downregulation in poplar results in the incorporation of 5-hydroxyconiferyl alcohol into lignins via typical radical coupling reactions, but post-coupling quinone methide internal trapping reactions produce novel benzodioxane units in the lignin. CAD (cinnamyl alcohol dehydrogenase) downregulation results in the incorporation of the hydroxycinnamyl aldehyde monolignol precursors intimately into the polymer. Sinapyl aldehyde cross-couples 8-O-4 with both guaiacyl and syringyl units in the growing polymer, whereas coniferyl aldehyde cross-couples 8-O-4 only with syringyl units, reflecting simple chemical cross-coupling propensities. The incorporation of hydroxycinnamyl aldehyde and 5-hydroxyconiferyl alcohol monomers indicates that these monolignol intermediates are secreted to the cell wall for lignification. The recognition that novel units can incorporate into lignins portends significantly expanded opportunities for engineering the composition and consequent properties of lignin for improved utilization of valuable plant resources.

Alcohol Oxidoreductases↗

PubMed2001

NMR evidence for benzodioxane structures resulting from incorporation of 5-hydroxyconiferyl alcohol into Lignins of O-methyltransferase-deficient poplars.

Benzodioxane structures are produced in lignins of transgenic poplar plants deficient in COMT, anO-methyltransferase required to produce lignin syringyl units. They result from incorporation of 5-hydroxyconiferyl alcohol into the monomer supply and confirm that phenols other than the three traditional monolignols can be integrated into plant lignins.

Dioxanes↗

PubMed2000

Diverse effects of overexpression of LEAFY and PTLF, a poplar (Populus) homolog of LEAFY/FLORICAULA, in transgenic poplar and Arabidopsis.

PTLF, the Populus trichocarpa homolog of LEAFY (LFY) and FLORICAULA, was cloned to assess its function in a dioecious tree species. In situ hybridization studies showed that the gene was expressed most strongly in developing inflorescences. Expression was also seen in leaf primordia and very young leaves, most notably in apical vegetative buds near inflorescences, but also in seedlings. Although ectopic expression of the PTLF cDNA in Arabidopsis accelerated flowering, only one of the many tested transgenic lines of Populus flowered precociously. The majority of trees within a population of 3-year-old transgenic hybrid Populus lines with PTLF constitutively expressed showed few differences when compared to controls. However, phenotypic effects on growth rate and crown development, but not flowering, were seen in some trees with strong PTLF expression and became manifest only as the trees aged. Competence to respond to overexpression of LFY varied widely among Populus genotypes, giving consistent early flowering in only a single male P. tremula x P. tremuloides hybrid and causing gender change in another hybrid genotype. PTLF activity appears to be subject to regulation that does not affect heterologously expressed LFY, and is dependent upon tree maturation. Both genes provide tools for probing the mechanisms of delayed competence to flower in woody plants.

Amino Acid Sequence↗

PubMed1999

Structural alterations of lignins in transgenic poplars with depressed cinnamyl alcohol dehydrogenase or caffeic acid O-methyltransferase activity have an opposite impact on the efficiency of industrial kraft pulping

We evaluated lignin profiles and pulping performances of 2-year-old transgenic poplar (Populus tremula x Populus alba) lines severely altered in the expression of caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) or cinnamyl alcohol dehydrogenase (CAD). Transgenic poplars with CAD or COMT antisense constructs showed growth similar to control trees. CAD down-regulated poplars displayed a red coloration mainly in the outer xylem. A 90% lower COMT activity did not change lignin content but dramatically increased the frequency of guaiacyl units and resistant biphenyl linkages in lignin. This alteration severely lowered the efficiency of kraft pulping. The Klason lignin level of CAD-transformed poplars was slightly lower than that of the control. Whereas CAD down-regulation did not change the frequency of labile ether bonds or guaiacyl units in lignin, it increased the proportion of syringaldehyde and diarylpropane structures and, more importantly with regard to kraft pulping, of free phenolic groups in lignin. In the most depressed line, ASCAD21, a substantially higher content in free phenolic units facilitated lignin solubilization and fragmentation during kraft pulping. These results point the way to genetic modification of lignin structure to improve wood quality for the pulp industry.

Journal Article↗

PubMed1995

Tissue- and cell-specific expression of a cinnamyl alcohol dehydrogenase promoter in transgenic poplar plants.

Cinnamyl alcohol dehydrogenase (CAD) which catalyses the synthesis of the cinnamyl alcohols, the immediate precursors of lignins, from the corresponding cinnamaldehydes is considered to be a highly specific marker for lignification. We have isolated and characterized a CAD genomic clone from eucalyptus, a woody species of economic importance. The full-length promoter (EuCAD, 2.5 kb) and a series of 5' deletions were fused to the beta-glucuronidase (GUS) reporter gene. These constructs were tested in a homologous transient expression system of eucalyptus protoplasts which enabled the identification of several regions involved in transcriptional control. In order to study the spatial and developmental regulation of the CAD gene, the chimeric gene fusion (EuCAD-GUS) was then transferred via Agrobacterium tumefaciens-mediated transformation into poplar, an easily transformable woody angiosperm. Quantitative fluorometric assays conducted on eight independent in vitro transformants showed that GUS activity was highest in roots followed thereafter by stems and leaves. Histochemical staining for GUS activity on both in vitro primary transformants and more mature greenhouse-grown plants indicated a specific expression in the vascular tissues of stems, roots, petioles and leaves. At the onset of xylem differentiation, GUS activity was detected in parenchyma cells differentiating between the xylem-conducting elements. After secondary growth has occurred, GUS activity was localized in xylem ray cells and parenchyma cells surrounding the lignified phloem and sclerenchyma fibers. This first characterization of a woody angiosperm CAD promoter provides functional evidence for the role of CAD in lignification and suggests that parenchyma cells expressing CAD may provide lignin precursors to the adjacent lignified elements (vessels and fibres).

Alcohol Oxidoreductases↗

PubMed1995

IS292: a novel insertion element from Agrobacterium.

A new insertion sequence, IS292, located in the 6b gene of a nopaline-type Agrobacterium strain (X88-292) isolated from poplar was identified and sequenced. IS292 is 2494 bp long, has 21 bp inverted terminal repeats with two mismatches, and generates 10 bp direct repeats upon integration. No sequence similarity was found between IS292 and other insertion elements associated with Agrobacterium, but it shows strong similarity with ISR/1 from Rhizobium leguminosarum bv. viciae. The occurrence of IS292-like sequences in various Agrobacterium isolates, especially different Agrobacterium strains isolated from the same biotope, was demonstrated by DNA hybridization.

Amino Acid Sequence↗

PubMed1994

Enhancement of the endogenous cytokinin concentration in poplar by transformation with Agrobacterium T-DNA gene ipt.

The agrobacterial isopentenyltransferase (ipt) gene, under the control of its native promoter, was transferred to poplar (Populus tremula x P. alba) by an Agrobacterium co-cultivation method. The ipt-transformed stem explants developed calli that regenerated many buds in the absence of exogenous cytokinins. Microcuttings of the ipt transformants exhibited frequently branching shoots with short internodes that were unable to root. In this material, the concentrations of zeatin, zeatin riboside and isopentenyladenosine, determined by an indirect enzyme-linked immunosorbent assay (ELISA), were 4.8-. 17.1- and 14.6-fold higher, respectively, than in non-transformed shoots. Results are discussed with regard to cytokinin metabolism.

Journal Article↗

PubMed1989

Hormone Levels and Apical Dominance in the Aquatic Fern Marsilea drummondii A. Br.

Terminal buds and successively subjacent lateral buds of the water fern, Marsilea drummondii, were examined to determine the pattern of hormone distribution in relation to apical dominance. Quantitative levels of indole-3-acetic acid (IAA), abscisic acid (ABA), zeatin and zeatin riboside (Z and ZR), and isopentenyladenosine (iPA) were determined by a solid-phase immunoassay using polycional antihormone antibodies. Enzyme-linked immunosorbent assay was used following a one-step HPLC purification procedure to obtain the free hormones. Active shoot apices contained the most IAA and Z-type cytokinins and inhibited buds the least. No significant differences in ABA levels were found leading to the conclusion that ABA did not play any role in apical dominance. The normal precedence of the most rapid outgrowth of the youngest inhibited bud as observed previously in decapitated plants was well correlated with its very high level of iPA observed in this study. The same phenomenon was observed in the median buds but with a weaker amplitude. The presence of this storage form could indicate that a bud at its entry into quiescence eventually looses the ability to hydroxylate iPA to Z-type cytokinins when it is fully inhibited. IAA and Z + ZR are concluded to be essential for lateral bud growth.

Journal Article↗

PubMed1987

An avidin-biotin solid phase ELISA for femtomole isopentenyladenine and isopentenyladenosine measurements in HPLC purified plant extracts.

A solid phase enzyme immunoassay was developed for isopentenyladenine (iP) and isopentenyladenosine (iPA) quantitation in HPLC purified plant extracts. It was performed on antigen-coated microtitration plates on which bound antibodies were indirectly labeled by the means of a biotinylated goat anti-rabbit antibody and an avidin-alkaline phosphatase conjugate. Less than 3 femtomoles of iP or iPA were easily detected and the measuring range extended from 3 femtomole to 1 picomole. The reproducibility has been tested and intra- and interassay variations did not exceed 5.0%. The specificity of iPA antibodies was good, as determined by cross-reactivity measurements with other adenylic compounds. The specificity of the measurements for iP and iPA was demonstrated by analysis of the immunoreactivity of fractions obtained by HPLC separation of a methanolic tobacco leaf extract.

Journal Article↗

PubMedDate not supplied

Expressed sequence tags from poplar wood tissues--a comparative analysis from multiple libraries.

Xylogenesis involves successive developmental processes--cambial division, cell expansion and differentiation, cell death--each occurring along a gradient from the cambium to the pith of the stem. Taking advantage of the high level of organisation of wood tissues, we isolated cambial zone (CZ), differentiating xylem (DX) and mature xylem (MX) from both tension wood (TW) and opposite wood (OW) of bent poplars. Four different cDNA libraries were then constructed and used to generate 10,062 EST, reflecting the genes expressed in the different wood tissues. For the most abundant clusters, the EST distributions were compared between libraries in order to identify genes specific or over-represented at some specific developmental stages. They clearly showed a developmental shift between CZ and DX, whereas there is a continuity of development between DX and MX. CZ was mainly characterized by clusters of genes involved in cell cycle, protein synthesis and fate. Interestingly, two clusters with no assigned function were found specific to the cambial zone. In DX and MX, clusters were mostly involved in methylation of lignin precursors and microtubule cytoskeleton. In addition, in DX, EST from TW and OW were compared: five clusters of arabinogalactan proteins, one for sucrose synthase and one for fructokinase were specific or over-represented in TW. Moreover, a putative transcription factor and a cluster of unknown function were also identified in DX-TW. The informative comparison of multiple libraries prepared from wood tissues led to the identification of genes--some with still unknown functions--putatively involved in xylogenesis and tension wood formation.

Cell Cycle↗