Cell suspension cultures of Zeamays L. were adapted to grow under conditions of NaCl stress, which increased the cell‐wall pectin content of these cells by 31% compared with unadapted cells (controls). Both cultures were treated for 5 or 10 min with pectin methylesterase (PME) and afterwards incubated in the presence of Al for 2 h. The different capabilities of the cells to synthesise callose due to pre‐treatment were taken into account by calculating relative Al‐induced callose induction (digitonin=100%). Only in salt‐adapted cells with a degree of methylation of cell‐wall pectin (DM) decreasing from 34% (control) to 13%, did PME treatment enhance total and BaCl2‐non‐exchangeable Al contents and Al sensitivity as indicated by increased callose formation. In a further step, a wider variation in DM was achieved by subculturing the NaCl‐adapted cells for up to 3 weeks without NaCl supply and adapting them to the cellulose‐synthesis inhibitor 2,6‐dichlorbenzonitrile (DCB). This reduced DM to 26%, while short‐term treatment with pectolyase resulted in the lowest DM (12%). After the 2 h Al treatment, there was a close negative relationship between DM and relative callose formation of Al contents, with the exception of pectolyase‐treated cells. In addition, intact plants of Solanumtuberosum L. genotypes were characterised for their Al sensitivity in hydroponics using root elongation, Al‐induced callose formation and Al contents of root tips as parameters. Based on all three parameters, the transgenic potato mutant overexpressing PME proved to be more Al‐sensitive than the wild type, the Al‐resistant and even the Al‐sensitive potato cultivar. Especially in the root tips (1 cm), Al treatment (2 h, 50 μM) increased the activity of PME more in the Al‐sensitive than in the Al‐resistant genotypes. The presented data emphasise the importance of the DM of the pectin matrix and the activity of PME for the expression of Al toxicity and Al resistance.

Transgenic potato (Solanum tuberosum L.) plants were constructed with a Petunia in¯ata-derived cDNA encoding a pectin methyl esterase (PME; EC 3.1.1.11) in sense orientation under the control of the cauli¯ower mosaic virus 35S promoter. The PME activity was elevated in leaves and tubers of the transgenic lines but slightly reduced in apical segments of stems from mature plants. Stem segments from the base of juvenile PME-overexpressing plants did not dier in PME activity from the control, whereas in apical parts PME was less active than in the wild-type. During the early stages of development stems of these trangenic plants elongated more rapidly than those of the wild-type. Further evidence that overexpression of a plant-derived PME has an impact on plant development is based on modi®cations of tuber yield, which was reduced in the transgenic lines. Cell walls from transgenic tubers showed signi®cant dierences in their cation-binding properties in comparison with the wildtype. In particular, cell walls displayed increased anity for sodium and calcium, while potassium binding was constant. Furthermore, the total ion content of transgenic potatoes was modi®ed. Indications of PMEmediated dierences in the distribution of ions in transgenic plants were also obtained by monitoring relaxations of the membrane potential of roots subsequent to changes in the ionic composition of the bathing solution. However, no eects on the chemical structure of pectin from tuber cell walls could be detected.

Two pectin methyl esterases (PMEs; EC 3.1.1.11) from Solanum tuberosum were isolated and their expression characterised. One partial clone ( pest1) was expressed in leaves and fruit tissue, while pest2 was a functional full-length clone and was expressed ubiquitously, with a preference for aerial organs. Potato plants were transformed with a chimeric antisense construct that was designed to simultaneously inhibit pest1 and pest2 transcript accumulation; however, reduction of mRNA levels was confined to pest2. The decrease in pest2 transcript was accompanied by up to 50% inhibition of total PME activity, which was probably due to the reduction of only one PME isoform. PME inhibition affected plant development as reflected by smaller stem elongation rates of selected transformants when compared with control plants, leading to a reduction in height throughout the entire course of development. Expansion rates of young developing leaves were measured simultaneously by two displacement transducers in the direction of the leaf tip (proximal-distal axis) and in the perpendicular direction (medial-lateral axis). Significant differences in leaf growth patterns were detected between wild-type and transgenic plants. We suggest that these visual phenotypes could be correlated with modifications of ion accumulation and partitioning within the transgenic plants. The ion-binding capacities of cell walls from PME-inhibited plants were specifically modified as they preferentially bound more sodium, but less potassium and calcium. X-ray microanalysis also indicated an increase in the concentration of several ions within the leaf apoplast of transgenic plants. Moreover, quantification of the total content of major cations revealed differences specific for a given element between the leaves of PME-inhibited and wild-type plants. Reduced growth rates might also be due to effects of PME inhibition on pectin metabolism, predominantly illustrated by an accumulation of galacturonic acid over other cell wall components.

Lipochitin oligosaccharides are organogenesis-inducing signal molecules produced by rhizobia to establish the formation of nitrogen-fixing root nodules in leguminous plants. Chitin oligosaccharide biosynthesis by the Mesorhizobium loti nodulation protein NodC was studied in vitro using membrane fractions of an Escherichia coli strain expressing the cloned M. loti nodC gene. The results indicate that prenylpyrophosphate-linked intermediates are not involved in the chitin oligosaccharide synthesis pathway. We observed that, in addition to N-acetylglucosamine (GlcNAc) from UDP-GlcNAc, NodC also directly incorporates free GlcNAc into chitin oligosaccharides. Further analysis showed that free GlcNAc is used as a primer that is elongated at the nonreducing terminus. The synthetic glycoside p-nitrophenyl-beta-N-acetylglucosaminide (pNPGlcNAc) has a free hydroxyl group at C4 but not at C1 and could also be used as an acceptor by NodC, confirming that chain elongation by NodC takes place at the nonreducing-terminal residue. The use of artificial glycosyl acceptors such as pNPGlcNAc has not previously been described for a processive glycosyltransferase. Using this method, we show that also the DG42-directed chitin oligosaccharide synthase activity, present in extracts of zebrafish embryos, is able to initiate chitin oligosaccharide synthesis on pNPGlcNAc. Consequently, chain elongation in chitin oligosaccharide synthesis by M. loti NodC and zebrafish DG42 occurs by the transfer of GlcNAc residues from UDP-GlcNAc to O4 of the nonreducing-terminal residue, in contrast to earlier models on the mechanism of processive beta-glycosyltransferase reactions.

Synthesis of chitin oligosaccharides by NodC is the first committed step in the biosynthesis of rhizobial lipochitin oligosaccharides (LCOs). The distribution of oligosaccharide chain lengths in LCOs differs between variousRhizobium species. We expressed the cloned nodC genes of Rhizobium meliloti, R. leguminosarum bv. viciae, and R. loti in Escherichia coli. The in vivo activities of the various NodC proteins differed with respect to the length of the major chitin oligosaccharide produced. The clearest difference was observed between strains with R. meliloti and R. loti NodC, producing chitintetraose and chitinpentaose, respectively. In vitro experiments, using UDP-[ 14 C]GlcNAc as a precursor, show that this difference reflects intrinsic properties of these NodC proteins and that it is not influenced by the UDP-GlcNAc concentration. Analysis of oligosaccharide chain lengths in LCOs produced by a R. leguminosarum bv. viciae nodC mutant, expressing the three cloned nodC genes mentioned above, shows that the difference in oligosaccharide chain length in LCOs of R. meliloti and R. leguminosarum bv. viciae is due only to nodC. The exclusive production of LCOs which contain a chitinpentaose backbone by R. loti strains is not due to NodC but to end product selection by Nod proteins involved in further modification of the chitin oligosaccharide. These results indicate that nodC contributes to the host specificity of R. meliloti, a conclusion consistent with the results of several studies which have shown that the lengths of the oligosaccharide backbones of LCOs can strongly influence their activities on host plants.Bacteria belonging to the genera Rhizobium, Azorhizobium, and Bradyrhizobium are able to induce the formation of a new organ, a nodule, on the roots of leguminous plants. The synthesis of signal molecules by these members of the family Rhizobiaceae is essential for this nodulation process (reviewed in references 3, 6, and 29). These signal molecules consist of an oligomer of ␤134-linked N-acetyl-D-glucosamine (GlcNAc) residues which is N-acylated at the nonreducing residue and hence are designated lipochitin oligosaccharides (LCOs). The structures of LCOs produced by different rhizobia vary in (i) the presence of additional groups on either the reducing or nonreducing terminus of the chitin oligosaccharide, (ii) the type of acyl chain present on the nonreducing residue, and (iii) the length of the oligosaccharide backbone. The presence of special, highly unsaturated fatty acids as well as of additional substitutions on the sugar backbone has been shown to play a crucial role in the determination of the host specificity of nodulation (recently reviewed in references 26 and 30). Several studies have also shown that the length of the oligosaccharide backbone of LCOs can strongly influence their activity on host plants (1,8,11,27,35). The chain length of the major oligosaccharide moiety in LCOs differs most between LCOs produced by Rhizobium meliloti and R. loti. R. meliloti LCOs mainly contain a chitintetraose...