Polyploidy, the presence of more than two complete sets of chromosomes in an organism, has significantly shaped the genomes of angiosperms during evolution. Two forms of polyploidy are often considered: allopolyploidy, which originates from interspecies hybrids, and autopolyploidy, which originates from intraspecies genome duplication events. Besides affecting genome organization, polyploidy generates other genetic effects. Synthetic allopolyploid plants exhibit considerable transcriptome alterations, part of which are likely caused by the reunion of previously diverged regulatory hierarchies. In contrast, autopolyploids have relatively uniform genomes, suggesting lower alteration of gene expression. To evaluate the impact of intraspecies genome duplication on the transcriptome, we generated a series of unique Arabidopsis thaliana autotetraploids by using different ecotypes. A. thaliana autotetraploids show transcriptome alterations that strongly depend on their parental genome composition and include changed expression of both new genes and gene groups previously described from allopolyploid Arabidopsis . Alterations in gene expression are stable, nonstochastic, developmentally specific, and associated with changes in DNA methylation. We propose that Arabidopsis possesses an inherent and heritable ability to sense and respond to elevated, yet balanced chromosome numbers. The impact of natural variation on alteration of autotetraploid gene expression stresses its potential importance in the evolution and breeding of plants.

SummaryWe previously showed that H-2Ka-restricted cytotoxic T lymphocyte (CTL) clones specific for a single nonapeptide derived from the Plasraodium berghei circumsporozoite (PbCS) protein displayed T cell receptors (TCRs) of highly diverse primary structure. We have now analyzed the TCR repertoire of CTLs that recognize a peptide derived from the human dass I major histocompatibility complex (MHC) molecule HLA-Cw3 in association with the same murine class I MHC molecule H-2K a. We first sequenced the TCR o~ and/3 genes of the CTL clone Cw3/1.1 and, based on this genomic analysis, the TCR c~ and/3 cDNA junctional regions of 23 independent H-2K drestricted CTL clones specific for HLA-Cw3. The results show that the TCR chains display very limited heterogeneity, both in terms of Vet, Jo~, V~ and J/3 segments, and in terms of length and sequence of the CDR3 cx and/3 loops. The TCR repertoire used in vivo was then analyzed by harvesting CTL populations from the peritoneal cavity of immune mice. The peritoneal exudate lymphocytes (PELs) displayed HLA-Cw3-specific cytolytic activity in the absence of any stimulation in vitro. Remarkably, most of these freshly isolated PELs expressed TCRs that shared the same structural features as those from HLA-Cw3-reactive CTL clones. Thus, our results show that a peptide from HLA-Cw3 presented by H-2K d selects CTLs that bear TCILs of very limited diversity in vivo. When taken together with the high diversity of the TCRs specific for the PbCS peptide, these findings suggest that natural tolerance to self peptides presented by class I MHC molecules may substantially reduce the size of the TCR repertoire of CTLs specific for antigenic peptides homologous to self.

Deficiency of the essential nutrient boron (B) in the soil is one of the most widespread micronutrient deficiencies worldwide, leading to developmental defects in root and shoot tissues of plants, and severe yield reductions in many crops. Despite this agricultural importance, the underlying mechanisms of how B shapes plant developmental and morphological processes are still not unequivocally understood in detail. This review evaluates experimental approaches that address our current understanding of how B influences plant morphological processes by focusing on developmental defects observed under B deficiency. We assess what is known about mechanisms that control B homeostasis and specifically highlight: (i) limitations in the methodology that is used to induce B deficiency; (ii) differences between mutant phenotypes and normal plants grown under B deficiency; and (iii) recent research on analyzing interactions between B and phytohormones. Our analysis highlights the need for standardized methodology to evaluate the roles of B in the cell wall versus other parts of the cell.

Mycoplasma arthritidis produces an as yet undefined soluble molecule (MAS) that has a potent mitogenic effect on T cells of several species. We have used cloned human cytotoxic and proliferative T lymphocytes to dissect the molecular mechanism of T cell activation by this mitogen. Reactivity to MAS is clonally expressed among T cell receptor (TcR) alpha/beta chain-expressing T cell clones of CD4+ or CD8+ phenotype, as well as CD4-8- TcR alpha/beta chain-negative T lymphocyte clones expressing the CD3-associated TcR gamma chain. MAS is able to induce cytotoxicity and/or proliferation in these T cell clones. For triggering of these T cells, regardless of their phenotype of specificity, the presence of autologous, allogeneic or xenogeneic major histocompatibility complex (MHC) class II molecules on accessory cells or target cells is necessary. However, T cells do not immunologically recognize MAS on class II molecules, since a direct action of MAS on the T cells themselves can be demonstrated. Triggering of T cells by MAS can be blocked by monoclonal antibodies against CD2, CD3 and the TcR alpha/beta chain dimer. We discuss as a possible explanation that MAS is a functionally bivalent molecule cross-linking TcR and MHC class II molecules. Thus, the mechanism of T cell activation by MAS has striking similarities to the mechanisms by which Staphylococcal enterotoxins activate T cells. It is intriguing that a similar mitogenic principle has been developed by two evolutionary distinct pathogenic microorganisms.

We have characterized a panel of monoclonal antibodies (mAb) produced by immunizing rats with two distinct soluble mouse alpha/beta T cell receptor (TcR). Fifty mAb were found to react with the corresponding surface-bound TcR. Such observations suggest that the soluble TcR molecules used as immunogen are folded in a conformation similar to the native structure. Furthermore, the binding to T cells of four antibodies was found to correlate with the expression of the V alpha 8, V beta 2 or V beta 10 gene segments. Finally, staining of T lymphocytes from various mouse strains suggests that (a) the two anti-V alpha 8 antibodies recognize different epitopes, and each on only a fraction of V alpha 8+ cells; (b) the anti-V beta 10 mAb identifies a V beta 10 polymorphism among mouse strains, and (c) T cells expressing the V beta 2 or V beta 10 gene segments are not subject to major clonal deletion events induced by the major histocompatibility complex class II and Mls products which were tested.

Technical University Hamburg-Harburg, Hamburg, Germany 4-Hydroxybenzoate hydroxylase from Pseudomonus sp. CBS3 was purified by five consecutive steps to apparent homogeneity. The enrichment was 50-fold with a yield of about 20%. The enzyme is a homodimeric flavoprotein monooxygenase with each 44-kDa polypeptide chain containing one FAD molecule as a rather weakly bound prosthetic group. In contrast to other 4-hydroxybenzoate hydroxylases of known primary structure, the enzyme preferred NADH over NADPH as electron donor. The pH optimum for catalysis was pH 8.0 with a maximum turnover rate around 45°C. Chloride ions were inhibitory, and competitive with respect to NADH.4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 has a narrow substrate specificity. In addition to the transformation of 4-hydroxybenzoate to 3,4-dihydroxybenzoate, the enzyme converted 2-tluoro-4-hydroxybenzoate, 2-cliloro-4-hydroxybenzoate, and 2,4-dihydroxybenzoate. With all aromatic substrates, no uncoupling of hydroxylation was observed.The gene encoding 4-hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 was cloned in Escherichia coli. Nucleotide sequence analysis revealed an open reading frame of 11 82 bp that corresponded to a protein of 394 amino acid residues. Upstream of the pobA gene, a sequence resembling an E. coli promotor was identified, which led to constitutive expression of the cloned gene in E. coli TG1 . The deduced amino acid sequence of Pseudomanus sp. CBS3 4-hydroxybenzoate hydroxylase revealed 53 % identity with that of the pobA enzyme from Pseudomonasfluorescens for which a three-dimensional structure is known. The active-site residues and the fingerprint sequences associated with FAD binding are strictly conserved. This and the conservation of secondary structures implies that the enzymes share a similar three-dimensional fold. Based on an isolated region of sequence divergence and site-directed mutagenesis data of 4-hydroxybenzoate hydroxylase from f? ,fluorescens, it is proposed that helix H2 is involved in determining the coenzyme specificity.

Several observations suggest that the micronutrient boron (B) has a stabilising role in the plasma membrane (PM), supporting functions in PM-linked (hormone) signalling processes. However, this role is poorly characterised. Here we show treatment with boronic acids, specific competitors of B, phenocopies the Arabidopsis thaliana rootless pattern mutant monopteros. At least in part, this is caused by phenylboronic acid (PBA)-induced internalisation of the membranelocalised auxin efflux carrier PINFORMED1 (PIN1) in the early embryo. PIN1 internalisation interrupts the feedback signal transduction cascade involving the phytohormone auxin, PIN1 and the transcription factor gene MONOPTEROS. This entails several effects, including abnormal development of vascular cell precursors, suppression of MONOPTEROS downstream targets and loss of the root auxin maximum -essential signals for root meristem development. While PIN1 is internalised, we observe a differential effect of PBA on other proteins, which are either unaffected, internalised or, as in the case of the B transporter BOR1, stabilised at the PM. These findings suggest a competition of PBA with B for plant membrane proteins and might shed light on the function of B at the PM.

The diversity of plant architecture is determined by axillary meristems (AMs). AMs are produced from small groups of stem cells in the axils of leaf primordia and generate vegetative branches and reproductive inflorescences. Previous studies identified genes critical for AM development that function in auxin biosynthesis, transport, and signaling. barren stalk1 (ba1), a basic helix-loop-helix transcription factor, acts downstream of auxin to control AM formation. Here, we report the cloning and characterization of barren stalk2 (ba2), a mutant that fails to produce ears and has fewer branches and spikelets in the tassel, indicating that ba2 functions in reproductive AM development. Furthermore, the ba2 mutation suppresses tiller growth in the teosinte branched1 mutant, indicating that ba2 also plays an essential role in vegetative AM development. The ba2 gene encodes a protein that co-localizes and heterodimerizes with BA1 in the nucleus. Characterization of the genetic interaction between ba2 and ba1 demonstrates that ba1 shows a gene dosage effect in ba2 mutants, providing further evidence that BA1 and BA2 act together in the same pathway. Characterization of the molecular and genetic interaction between ba2 and additional genes required for the regulation of ba1 further supports this finding. The ba1 and ba2 genes are orthologs of rice genes, LAX PANICLE1 (LAX1) and LAX2, respectively, hence providing insights into pathways controlling AMs development in grasses.