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Biomedical subjects

W Boerjan

Publications and source records attributed to W Boerjan.

34 records · Page 2Linked to original sources
PubMed1999

Evolution of plant defense mechanisms. Relationships of phenylcoumaran benzylic ether reductases to pinoresinol-lariciresinol and isoflavone reductases.

Pinoresinol-lariciresinol and isoflavone reductase classes are phylogenetically related, as is a third, the so-called "isoflavone reductase homologs." This study establishes the first known catalytic function for the latter, as being able to engender the NADPH-dependent reduction of phenylcoumaran benzylic ethers. Accordingly, all three reductase classes are involved in the biosynthesis of important and related phenylpropanoid-derived plant defense compounds. In this investigation, the phenylcoumaran benzylic ether reductase from the gymnosperm, Pinus taeda, was cloned, with the recombinant protein heterologously expressed in Escherichia coli. The purified enzyme reduces the benzylic ether functionalities of both dehydrodiconiferyl alcohol and dihydrodehydrodiconiferyl alcohol, with a higher affinity for the former, as measured by apparent Km and Vmax values and observed kinetic 3H-isotope effects. It abstracts the 4R-hydride of the required NADPH cofactor in a manner analogous to that of the pinoresinol-lariciresinol reductases and isoflavone reductases. A similar catalytic function was observed for the corresponding recombinant reductase whose gene was cloned from the angiosperm, Populus trichocarpa. Interestingly, both pinoresinol-lariciresinol reductases and isoflavone reductases catalyze enantiospecific conversions, whereas the phenylcoumaran benzylic ether reductase only shows regiospecific discrimination. A possible evolutionary relationship among the three reductase classes is proposed, based on the supposition that phenylcoumaran benzylic ether reductases represent the progenitors of pinoresinol-lariciresinol and isoflavone reductases.

Amino Acid Sequence↗
PubMed1999

Carpel, a new Arabidopsis epi-mutant of the SUPERMAN gene: phenotypic analysis and DNA methylation status.

The carpel (car) mutation affects the morphology of reproductive organs in Arabidopsis thaliana. car flowers have an increased number of carpels, on average 2.7 +/- 0.8 instead of two in the wild type. Through allelism test with fon1-3 and analysis of the methylation state of the SUPERMAN (SUP) gene in car mutants, we show that car is an epi-mutation of SUP. The methylation pattern of car is clearly distinct from that of fon1-3, another epi-mutation of the SUP gene. Methylation was found predominantly in Cp(A/T)p(A/G) triplets and in CpG pairs. We suggest that the extensive SUP methylation in car has arisen from an abundant methylation of a single CpG site that was already present in abscisic acid-insensitive (abi3-4) mutants, from which car was segregating.

Arabidopsis↗
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↗
PubMed1998

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. x 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.

Arabidopsis↗
PubMed1998

Purification and characterization of peroxidases correlated with lignification in poplar xylem.

Lignin is an integral cell wall component of all vascular plants. Peroxidases are widely believed to catalyze the last enzymatic step in the biosynthesis of lignin, the dehydrogenation of the p-coumaryl alcohols. As the first stage in identifying lignin-specific peroxidase isoenzymes, the classical anionic peroxidases found in the xylem of poplar (Populus trichocarpa Trichobel) were purified and characterized. Five different poplar xylem peroxidases (PXP 1, PXP 2, PXP 3-4, PXP 5, and PXP 6) were isolated. All five peroxidases were strongly glycosylated (3.6% to 4.9% N-glucosamine), with apparent molecular masses between 46 and 54 kD and pI values between pH 3.1 and 3.8. Two of the five isolated peroxidases (PXP 3-4 and PXP 5) could oxidize the lignin monomer analog syringaldazine, an activity previously correlated with lignification in poplar. Because these isoenzymes were specifically or preferentially expressed in xylem, PXP 3-4 and PXP 5 are suggested to be involved in lignin polymerization.

Amino Acid Sequence↗
PubMed1996

Transgenic Arabidopsis tester lines with dominant marker genes.

The map positions of a set of eight T-DNA insertions in the Arabidopsis genome have been determined by using closely linked visible markers. The insertions are dispersed over four of the five chromosomes. Each T-DNA insert contains one or more of the chimeric marker genes neomycin phosphotransferase (neo), hygromycin phosphotransferase (hpt), phosphinothricin acetyltransferase (bar), beta-glucuronidase (gusA) and indole-3-acetamide hydrolase (iaaH). The neo, hpt and bar marker genes are dominant in a selective germination assay or when used as DNA markers in a polymerase chain reaction. These dominant markers will allow recombinants to be discerned in a germinating F2 population, one generation earlier than with a conventional recessive marker. The transgenic marker lines will speed up and simplify the isolation of recombinants in small genetic intervals, a rate-limiting step in positional cloning strategies. The transgenic lines containing the hpt marker will also be of interest for the isolation of deletion mutants at the T-DNA integration sites.

Acetyltransferases↗
PubMed1996

Tissue-specific expression conferred by the S-adenosyl-L-methionine synthetase promoter of Arabidopsis thaliana in transgenic poplar.

In Arabidopsis the promoter of the gene encoding S-adenosyl-L-methionine synthetase (SAM-S) Psam-1 confers expression preferentially in the vascular tissue. In search for promoters that drive expression in particular cells of the lignifying tissues in trees, we have analyzed the expression pattern conferred by the Psam-1 promoter in transgenic poplar. Histochemical analyses demonstrated beta-glucuronidase (GUS) activity mainly in phloem and cortex tissue throughout the plant, and in root tips. Fluorimetric assays showed high GUS activity in the tissues outside (phloem, cortex and cork) compared to those inside (xylem and pith) of the cambial layer. In contrast, the endogenous SAM-S activity was high in tissues inside and low in tissues outside of the cambial layer. RNA gel blot analysis demonstrated a high transcript level of the endogenous sam-s gene(s) in tissues both outside and inside the cambial layer. This indicates that the low SAM-S activity in the bark was at least partially due to translational and/or post-translational regulation of the endogenous sam-s gene(s). In dormant transgenics, the tissue specificity was conserved, but the activity levels were up to 10-fold reduced.

Arabidopsis↗
PubMed1995

Superroot, a recessive mutation in Arabidopsis, confers auxin overproduction.

We have isolated seven allelic recessive Arabidopsis mutants, designated superroot (sur1-1 to sur1-7), displaying several abnormalities reminiscent of auxin effects. These characteristics include small and epinastic cotyledons, an elongated hypocotyl in which the connection between the stele and cortical and epidermal cells disintegrates, the development of excess adventitious and lateral roots, a reduced number of leaves, and the absence of an inflorescence. When germinated in the dark, sur1 mutants did not develop the apical hook characteristic of etiolated seedlings. We were able to phenocopy the Sur1- phenotype by supplying auxin to wild-type seedlings, to propagate sur1 explants on phytohormone-deficient medium, and to regenerate shoots from these explants by the addition of cytokinins alone to the culture medium. Analysis by gas chromatography coupled to mass spectrometry indicated increased levels of both free and conjugated indole-3-acetic acid. sur1 was crossed to the mutant axr2 and the altered-auxin response mutant ctr1. The phenotype of both double mutants was additive. The sur1 gene was mapped on chromosome 2 at 0.5 centimorgans from the gene encoding phytochrome B.

Arabidopsis↗
PubMed1994

Distinct phenotypes generated by overexpression and suppression of S-adenosyl-L-methionine synthetase reveal developmental patterns of gene silencing in tobacco.

S-Adenosyl-L-methionine synthetase (SAM-S) catalyzes the conversion of L-methionine and ATP into S-adenosyl-L-methionine. Tobacco plants that were transformed with a construct allowing high transcription levels of an Arabidopsis sam-s gene could be grouped into two main classes based on their morphology. One class developed yellow-green leaves and had high SAM-S activity and transgene mRNA levels, whereas the other class was stunted and had leather-like leaves, very low SAM-S activity, and suppressed mRNA level of the transgene. Because both overexpression and silencing of transgene expression led to distinct, abnormal phenotypes, the developmental pattern of transgene silencing was visualized. In the lower leaves, the suppressed phenotype was associated with the veins. In successive leaves, the area of the suppressed tissue increased until all newly developed leaves displayed the suppressed phenotype. In this study, a hypothesis is presented for this developmental gene silencing. Furthermore, transgenic plants with suppressed SAM-S activity had a characteristic smell, a consequence of the accumulation of L-methionine that is converted into the volatile methanethiol.

Amino Acids↗
PubMed1992

A new bioassay for auxins and cytokinins.

The authors have developed a sensitive bioassay that can be used to detect auxins as well as cytokinins. The bioassay is based on the expression in transformed tobacco (Nicotiana tabacum) mesophyll protoplasts of a chimeric gene, consisting of the upstream sequences of the Agrobacterium tumefaciens gene 5, coupled to the coding sequence of the beta-glucuronidase. The expression of this gene is induced by the presence of both auxin and cytokinin in the culture medium. Using this assay, indole-3-acetic acid was detected at 5 x 10(-8) molar, whereas trans-zeatin could be detected at 5 x 10(-11) molar. The assay can be performed in microtiter plates, allowing numerous samples to be analyzed simultaneously. Only 2.5 x 10(5) protoplasts are required for one individual assay in 250 microliters of culture medium and for qualitative results, the reaction is readily visualized by ultraviolet light.

Journal Article↗
PubMed1992

Molecular genetic approaches to plant development.

Higher plant morphogenesis has received renewed interest over the past few years. The improvement of molecular genetic approaches to generate tagged developmental mutants, for instance by T-DNA insertion, facilitated the isolation and characterization of the altered genes. Here we present recent progress on flower and root morphogenesis in the small crucifer Arabidopsis thaliana. The current model of Arabidopsis flower development is presented. We report on FLOWER1 (Fl1), which is a T-DNA-tagged ap2 allele. Our observations indicate that this Fl1 mutant has, besides the homeotic Ap2 phenotype, an aberrant seed coat, suggesting that this gene has also a function late in flower development. Furthermore, we present a brief summary about root development and focus on the super root (Sur) mutant, which is an ethyl methanesulfonate-induced mutant that produces excess lateral roots. Root explants of the Sur mutant, that do not develop further than the 4-leaf stage, can be induced to produce normal-looking shoots and flowers by addition of only cytokinin to the medium. The phenotype of Sur and its relation to the action of phytohormones is discussed.

Morphogenesis↗
PubMed1991

A novel seed protein gene from Vicia faba is developmentally regulated in transgenic tobacco and Arabidopsis plants.

We have isolated a novel gene, denoted USP, from Vicia faba var. minor, which corresponds to the most abundant mRNA present in cotyledons during early seed development; however, the corresponding protein does not accumulate in cotyledons. The characterized USP gene with its two introns is 1 of about 15 members of a gene family. A fragment comprising 637 bp of 5' flanking sequence and the total 5' untranslated region was shown to be sufficient to drive the mainly seed-specific expression of two reporter genes, coding for neomycin phosphotransferase II and beta-glucuronidase, in transgenic Arabidopsis thaliana and Nicotiana tabacum plants. We showed that the USP promoter becomes active in transgenic tobacco seeds in both the embryo and the endosperm, whereas its activity in Arabidopsis is detectable only in the embryo. Moreover, we demonstrated a transient activity pattern of the USP promoter in root tips of both transgenic host species.

Amino Acid Sequence↗
PubMed1991

Upstream sequences regulating legumin gene expression in heterologous transgenic plants.

We have previously isolated a legumin gene LeB4 from Vicia faba and shown that a 4.7 kb DNA fragment containing the gene leads to seed-specific expression in transgenic tobacco plants. Here we report that the 2.4 kb upstream sequence alone, when fused to either the neomycin phosphotransferase II (nptII) gene or the beta-glucuronidase (uidA) gene, leads to high enzyme levels in transgenic seeds of both tobacco and Arabidopsis. beta-Glucuronidase (GUS) activity is especially intense in the cotyledons fading out towards the embryonal root tip, a result confirmed by in situ hybridization. Staining of endosperm cells is consistent in both species. Analysis of a series of promoter deletion mutants fused to the nptII gene and introduced into tobacco plants revealed that about 1 kb of 5'-flanking sequence is sufficient for high-level expression but indirect evidence suggests the presence of weak positive regulatory elements further upstream. Deletions leaving only 0.2 kb of upstream sequence reduce enzyme levels to less than 10%. A deletion which destroys the legumin box with its seed protein gene-specific CATGCATG motif has no obvious effects on expression levels.

Blotting, Southern↗
PubMed1989

Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase.

S-Adenosylmethionine serves as a methyl group donor in numerous transmethylation reactions and plays a role in the biosynthesis of polyamines and ethylene. We have cloned and sequenced an S-adenosylmethionine synthetase gene (sam-1) of Arabidopsis thaliana. The deduced polypeptide sequence of the enzyme has extensive homology with the corresponding enzymes of Escherichia coli and yeast. Genomic hybridization indicates the presence of two adenosylmethionine synthetase genes per haploid Arabidopsis genome. RNA gel blot analysis shows that adenosylmethionine synthetase mRNA levels are high in stems and roots, correlating well with the higher enzyme activity in stems, compared with leaves. Histochemical analysis of transgenic Arabidopsis plants transformed with a chimeric beta-glucuronidase gene, under the control of 748-base pair 5' sequences of the sam-1 gene, demonstrates that the gene is expressed primarily in vascular tissues. In addition, high expression was observed in sclerenchyma and in the root cortex. A hypothesis for the strong cellular preference in the expression of the sam-1 gene is presented.

Amino Acid Sequence↗