Search PubMedSearch

Biomedical subjects

M Van Montagu

Publications and source records attributed to M Van Montagu.

At least 19 recordsLinked to original sources

Plant genomics.

The rapidity with which genomic sequences of the model plant Arabidopsis thaliana and soon of rice are becoming available has strongly boosted plant molecular biology research. Here, two main genomic fields will be discussed: the progress in different structural genome projects, such as mapping, sequencing, genome organization and comparative genomics, and the so-called functional genomics approaches to analyze the genome using such molecular tools as transcript profiling, micro-arrays, and insertional mutagenesis. In addition a section on bioinformatics is included.

DNA, Plant

A highly conserved kinase is an essential component for stress tolerance in yeast and plant cells.

Osmotic stress (drought, salt stress) is a major limiting factor for crop productivity in the world. Because cellular responses to osmotic stress are thought to be conserved in eukaryotes and because yeast is much more amenable than plants to genetic research, a functional strategy has been performed to identify limiting steps in osmotolerance of plants based on the complementation of yeast with a plant library. A new plant cDNA that encodes a functional homologue of the yeast Dbf2 kinase enhances salt, drought, cold, and heat tolerance upon overexpression in yeast as well as in transgenic plant cells.

Amino Acid Sequence

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

Mutational analysis of two Arabidopsis thaliana cyclin-dependent kinases in fission yeast.

We have analyzed five mutant alleles of two cyclin-dependent kinases from Arabidopsis thaliana, CDC2aAt and CDC2bAt, in Schizosaccharomyces pombe. Two of the five mutant alleles produced similar phenotypes for both cyclin-dependent kinases. The other three mutants caused phenotypes dependent on the particular cyclin-dependent kinase. Of all the mutant alleles, only two were found to possess a detectable kinase activity. Our mutational analysis lends further support for CDC2aAt being the true orthologue of the yeast cdc2. CDC2bAt, even though quite divergent from S. pombe cdc2, still retains the ability to interact with at least some essential cell cycle regulators, suggesting some functional homology with the yeast protein. Additionally, we demonstrated that the three amino acid deletion in the DL50 mutants results in the loss of the ability to interact with the suc1/CKS1 proteins.

Amino Acid Sequence

Evidence for an ancient chromosomal duplication in Arabidopsis thaliana by sequencing and analyzing a 400-kb contig at the APETALA2 locus on chromosome 4.

As part of the European Scientists Sequencing Arabidopsis program, a contiguous region (396607 bp) located on chromosome 4 around the APETALA2 gene was sequenced. Analysis of the sequence and comparison to public databases predicts 103 genes in this area, which represents a gene density of one gene per 3.85 kb. Almost half of the genes show no significant homology to known database entries. In addition, the first 45 kb of the contig, which covers 11 genes, is similar to a region on chromosome 2, as far as coding sequences are concerned. This observation indicates that ancient duplications of large pieces of DNA have occurred in Arabidopsis.

Arabidopsis

Classification of Arabidopsis thaliana gene sequences: clustering of coding sequences into two groups according to codon usage improves gene prediction.

While genomic sequences are accumulating, finding the location of the genes remains a major issue that can be solved only for about a half of them by homology searches. Prediction methods are thus required, but unfortunately are not fully satisfying. Most prediction methods implicitly assume a unique model for genes. This is an oversimplification as demonstrated by the possibility to group coding sequences into several classes in Escherichia coli and other genomes. As no classification existed for Arabidopsis thaliana, we classified genes according to the statistical features of their coding sequences. A clustering algorithm using a codon usage model was developed and applied to coding sequences from A. thaliana, E. coli, and a mixture of both. By using it, Arabidopsis sequences were clustered into two classes. The CU1 and CU2 classes differed essentially by the choice of pyrimidine bases at the codon silent sites: CU2 genes often use C whereas CU1 genes prefer T. This classification discriminated the Arabidopsis genes according to their expressiveness, highly expressed genes being clustered in CU2 and genes expected to have a lower expression, such as the regulatory genes, in CU1. The algorithm separated the sequences of the Escherichia-Arabidopsis mixed data set into five classes according to the species, except for one class. This mixed class contained 89 % Arabidopsis genes from CU1 and 11 % E. coli genes, mostly horizontally transferred. Interestingly, most genes encoding organelle-targeted proteins, except the photosynthetic and photoassimilatory ones, were clustered in CU1. By tailoring the GeneMark CDS prediction algorithm to the observed coding sequence classes, its quality of prediction was greatly improved. Similar improvement can be expected with other prediction systems.

Algorithms

PlantCARE, a plant cis-acting regulatory element database.

PlantCARE is a database of plant cis- acting regulatory elements, enhancers and repressors. Besides the transcription motifs found on a sequence, it also offers a link to the EMBL entry that contains the full gene sequence as well as a description of the conditions in which a motif becomes functional. The information on these sites is given by matrices, consensus and individual site sequences on particular genes, depending on the available information. PlantCARE is a relational database available via the web at the URL: http://sphinx.rug.ac.be:8080/PlantCARE/

Arabidopsis

Recombinant production of the p10CKS1At protein from Arabidopsis thaliana and 13C and 15N double-isotopic enrichment for NMR studies.

The CKS1At gene product, p10CKS1At from Arabidopsis thaliana, is a member of the cyclin-dependent kinase subunit (CKS) family of small proteins. These proteins bind the cyclin-dependent kinase (CDK)/cyclin complexes and play an essential, but still not precisely known role in cell cycle progression. To solve the structure of p10CKS1At, a protocol was needed to produce the quantity of protein large enough for nuclear magnetic resonance (NMR) spectroscopy. The first attempt to express CKS1At in Escherichia coli under the control of the T7 promoter was not successful. E. coli BL21(DE3) cotransformed with the CKS1At gene and the E. coli argU gene that encoded the arginine acceptor tRNAUCU produced a sufficient amount of p10CKS1At to start the structural study by NMR. Replacement of four rare codons in the CKS1At gene sequence, including a tandem arginine, by highly used codons in E. coli, restored also a high expression of the recombinant protein. Double-isotopic enrichment by 13C and 15N is reported that will facilitate the NMR study. Isotopically labeled p10CKS1At was purified to yield as much as 55 mg from 1 liter of minimal media by a two-step chromatographic procedure. Preliminary results of NMR spectroscopy demonstrate that a full structural analysis using triple-resonance spectra is feasible for the labeled p10CKS1At protein.

Amino Acid Sequence

Expression of CKS1At in Arabidopsis thaliana indicates a role for the protein in both the mitotic and the endoreduplication cycle.

Although endoreduplication is common in plants, little is known about the mechanisms regulating this process. Here, we report the patterns of endoreduplication at the cellular level in the shoot apex of Arabidopsis thaliana L. Heynh. plants grown under short-day conditions. We show that polyploidy is developmentally established in the pith, maturing leaves, and stipules. To investigate the role of the cell cycle genes CDC2aAt, CDC2bAt, CYCB1;1, and CKS1At in the process of endoreduplication, in-situ hybridizations were performed on the vegetative shoot apices. Expression of CDC2aAt, CDC2bAt, and CYCB1;1 was restricted to mitotically dividing cells. In contrast, CKS1At expression was present in both mitotic and endoreduplicating tissues. Our data indicate that CDC2aAt, CDC2bAt, and CYCB1;1 only operate during mitotic divisions, whereas CKS1At may play a role in both the mitotic and endoreduplication cycle.

Arabidopsis

AFLP analysis of genetic diversity within and between Arabidopsis thaliana ecotypes.

The degree of genetic diversity within and between 21 Arabidopsis thaliana (L.) Heynh ecotypes was estimated by AFLP analysis. Within seven of the 21 ecotypes, a low but significant level of polymorphism was detected, and for five of these ecotypes two or three distinct subgroups could be distinguished. As these ecotypes represent natural populations, this intraecotypic diversity reflects natural genetic variation and diversification within the ecotypes. The source of this diversity remains unclear but is intriguing in view of the predominantly self-fertilizing nature of Arabidopsis. Interrelationships between the different ecotypes were estimated after AFLP fingerprinting using two enzyme combinations (EcoRI/MseI and SacI/MseI) and a number of selective primer pairs. SacI recognition sites are less evenly distributed in the genome than EcoRI sites, and occur more frequently in coding sequences. In most cases, AFLP data from only one enzyme combination are used for genetic diversity analysis. Our results show that the use of two enzyme combinations can result in significantly different classifications of the ecotypes both in cluster and ordination analysis. This difference most probably reflects differences in the genomic distribution of the AFLP fragments generated, depending on the enzymes and selective primers used. For closely related varieties, as in the case of Arabidopsis ecotypes, this can preclude reliable classification.

Arabidopsis

Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility.

To improve the digestibility of the forage crop alfalfa (Medicago sativa L.), cinnamyl alcohol dehydrogenase (CAD), which catalyses the last step in the biosynthesis of the lignin monomers, was down-regulated by using an antisense approach. A subset of six transgenic lines with reduced CAD activity and control lines were analysed when grown in the greenhouse and in the field. The down-regulation of the CAD enzyme was associated with a red coloration of the stem. The lignin quantity remained unchanged, but the lignin composition, as determined by thioacidolysis, was altered. The highest reduction of CAD activity was associated with a lower syringyl/guaiacyl (S/G) ratio and a lower S+G yield, mainly because of a decreased amount of S units. An increase in in situ disappearance of dry matter and of cell wall residue was detected in one of the transgenic lines grown in the greenhouse, and for two of the lines grown in the field the rate of disappearance of dry matter slightly improved. Furthermore, these two lines had a higher solubility in alkali as shown by the lower yield of saponified residue. This study opens perspectives for improving forage crop digestibility by the modulation of enzymes involved in lignin biosynthesis.

Alcohol Oxidoreductases

Characterization of an Arabidopsis thaliana receptor-like protein kinase gene activated by oxidative stress and pathogen attack.

An Arabidopsis thaliana cDNA clone that encodes a putative receptor-like protein kinase gene (At-RLK3) was characterized. The deduced 667-amino acid protein consists of an amino-terminal signal sequence, an extracellular domain, a single transmembrane domain, and a cytoplasmic domain with characteristics of serine/threonine protein kinase. Because of the original features of its extracellular domain, the At-RLK3 protein is a member of a new class of receptor-like protein kinases. The At-RLK3 gene is present as a single copy within the Arabidopsis genome and its transcripts are detected in root, stem, leaf and flower. In cultured cells, the At-RLK3 gene is activated upon oxidative stress and salicylic acid treatment. In plants, the gene appears to be differentially regulated during various plant-pathogen interactions: upon inoculation with strains of Pseudomonas syringae pv. tomato harboring or not, different avr genes, At-RLK3 transcripts accumulate transiently at similar levels during both compatible and incompatible interactions. This gene is, however, preferentially expressed during the incompatible interaction induced by the soil-borne vascular bacteria, Ralstonia solanacearum. The involvement of At-RLK3 in signal transduction pathways during pathogen attack is discussed.

Amino Acid Sequence

High level accumulation of single-chain variable fragments in the cytosol of transgenic Petunia hybrida.

The accumulation of five murine single-chain variable fragments, binding to dihydroflavonol 4-reductase, was analyzed in transgenic Petunia hybrida plants. The five scFv-encoding sequences were cloned in an optimized plant transformation vector for expression in the cytosol under control of the 35S promoter. In a transient expression assay we found that the scFv expression levels were reproducible and correlated with those in stably transformed petunia. Our results show that accumulation in the cytosol strongly depends on the intrinsic properties of the scFv fragment. Three of the five scFv fragments accumulated to unexpectedly high levels in the cytosol of the primary transformants, but no phenotypic effect could be detected. Experimental results indicate that one of the scFv fragments accumulated in the cytosol to 1% of the total soluble protein as a functional antigen-binding protein in the absence of disulphide bonds. This observation supports the idea that certain antibody fragments do not need disulphide bonds to be stable and functional. Such scFv scaffolds provide new opportunities to design scFv fragments for immunomodulation in the cytosol.

Alcohol Oxidoreductases

Carbamoylation of azorhizobial Nod factors is mediated by NodU.

Lipochitooligosaccharides (LCOs) synthesized by Azorhizobium caulinodans ORS571 are substituted at the nonreducing-terminal residue with a 6-O-carbamoyl group. LCO biosynthesis in A. caulinodans is dependent on the nodABCSUIJZnoeC operon. Until now, the role of the nodulation protein NodU in the synthesis of azorhizobial LCOs remained unclear. Based on sequence similarities and structural analysis of LCOs produced by a nodU mutant, a complemented nodU mutant, and Escherichia coli DH5 alpha expressing the nodABCSU genes, NodU was shown to be involved in the carbamoylation step.

Amino Acid Sequence

Molecular markers and cell cycle inhibitors show the importance of cell cycle progression in nematode-induced galls and syncytia.

Root knot and cyst nematodes induce large multinucleated cells, designated giant cells and syncytia, respectively, in plant roots. We have used molecular markers to study cell cycle progression in these specialized feeding cells. In situ hybridization with two cyclin-dependent kinases and two cyclins showed that these genes were induced very early in galls and syncytia and that the feeding cells progressed through the G2 phase. By using cell cycle blockers, DNA synthesis and progression through the G2 phase, or mitosis, were shown to be essential for gall and syncytium establishment. When mitosis was blocked, further gall development was arrested. This result demonstrates that cycles of endoreduplication or other methods of DNA amplification are insufficient to drive giant cell expansion. On the other hand, syncytium development was much less affected by a mitotic block; however, syncytium expansion was inhibited.

Animals

Silencing of beta-1,3-glucanase genes in tobacco correlates with an increased abundance of RNA degradation intermediates.

Post-transcriptional gene silencing of beta-1,3 glucanase genes in the transgenic tobacco line T17 is characterised by an increased turnover and, as a consequence, reduced levels of gn1 transgene and endogenous beta-1,3 glucanase mRNAs. Here, additional gn1 RNAs, both larger and smaller than the full-length messenger, are shown to accumulate in silenced plants of the transgenic tobacco line T17. The longer-than-full-length gn1 RNAs are the result of cryptic processing of the gn1 messenger. The small gn1 RNAs in silenced plants correspond to distal and proximal parts of the mature gn1 messenger. The proximal RNA products are intact at their 5' extremity, but terminate at different positions at the 3'-end. The distal RNA products contain a poly(A) tail and are truncated to various positions at the 5'-end. These observations indicate that degradation of the mature gn1 transcript does not start at the 5'- or 3'-end, but rather are consistent with degradation of the gn1 transcript starting with an endonucleolytic cleavage followed by internal exonuclease digestion. Importantly, the truncated products are more abundant in silenced plants than in expressing plants. This suggests, together with the previously reported silencing-related increased gn1 mRNA turnover and the similar rates of gn1 transcription in silenced and expressing T17 plants, that the predominant decay route for the gn1 transcripts differs between silenced and expressing conditions.

Chromosome Mapping

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