Immunogold localization of trehalose-6-phosphate synthase in leaf segments of wild-type and transgenic tobacco plants expressing the AtTPS1 gene from Arabidopsis thaliana.

Following the establishment of a transgenic line of tobacco (B5H) expressing the trehalose-6-phosphate synthase (TPS) gene from Arabidopsis thaliana, a preliminary immunolocalization study was conducted using leaves of adequately watered B5H and wild-type plants. Immunocytochemical staining, followed by electron microscopy showed that the enzyme could be detected in both B5H and wild-type plants at two different levels. Quantification showed the signal to be two to three times higher in transgenic plants than in the wild type. This enzyme was markedly present in the vacuoles and the cell wall, and to a lesser extent in the cytosol. Moreover, a high profusion of gold particles was detected in adjacent cells and in the sieve elements. Occasional spots were also detected in chloroplasts and the nucleus, especially in the transgenic B5H line. No labeling signal was detected in mitochondria. Protein localization seems to confirm the important role of TPS in sugar metabolism and transport through the plant, which could explain its role in plant stress tolerance. Finally, it can be expected that TPS from tobacco has a relatively high similarity to the TPS of Arabidopsis thaliana.

Trehalose metabolism and glucose sensing in plants.

Plants sense and respond to changes in carbon and nitrogen metabolites during development and growth according to the internal needs of their metabolism. Sugar-sensing allows plants to switch off photosynthesis when carbohydrates are abundant. These processes involve regulation of gene and protein activity to allow plants the efficient use of energy storage. Besides being a key element in carbon metabolism, glucose (Glc) has unravelled as a primary messenger in signal transduction. It has been proved that hexokinase (HXK) is a Glc sensor. An unusual disaccharide named trehalose is present in very low levels in most plants except for the desiccation-tolerant plants known as 'resurrection' plants where trehalose functions as an osmoprotectant. We have shown that overexpression of the Arabidopsis trehalose-6-phosphate synthase gene (AtTPS1) in Arabidopsis promotes trehalose and trehalose-6-phosphate (T6P) accumulation. Seedlings expressing AtTPS1 displayed a Glc-insensitive phenotype. Transgenic lines germinated normally on Glc, in contrast to wild-type seedlings showing growth retardation and absence of chlorophyll and root elongation. Gene-expression analysis in transgenic plants showed up-regulation of several genes involved in sugar signalling and metabolism. These data suggest that AtTPS1 and accordingly T6P and trehalose play an important role in the regulation of Glc sensing and signalling genes during plant development.

An unexpected plethora of trehalose biosynthesis genes in Arabidopsis thaliana.

Trehalose accumulation has been documented in many organisms, such as bacteria and fungi, where it serves a storage and stress-protection role. Although conspicuously absent in most plants, trehalose biosynthesis genes were discovered recently in higher plants. We have uncovered a family of 11 TPS genes in Arabidopsis thaliana, one of which encodes a trehalose-6-phosphate (Tre6P) synthase, and a subfamily of which might encode the still elusive Tre6P phosphatases. A regulatory role in carbon metabolism is likely but might not be restricted to the TPS control of hexokinase activity as documented for yeast. Incompatibility between high trehalose levels and chaperone-assisted protein folding might be a reason why plants have evolved to accumulate some alternative stress-protection compounds to trehalose.

Localization and control of expression of Nt-Syr1, a tobacco SNARE protein.

Syntaxins and other SNARE proteins are crucial for intracellular vesicle trafficking, fusion and secretion. Previously, we isolated the syntaxin-related protein Nt-Syr1 from Nicotiana in a screen for ABA-related signalling elements, and demonstrated its role in determining the ABA sensitivity of stomatal guard cells. Because the location and expression of SNAREs are often important clues to their functioning, we have examined the distribution and stimulus-dependent expression of Nt-Syr1 between tissues, as well as its location within the cell, using antisera raised against purified recombinant peptides corresponding to overlapping cytosolic domains of Nt-Syr1. The Nt-Syr1 epitope was strongly represented in roots and to lesser extents in stems, leaves and flowers of well-watered plants. Biochemical analysis and examination of immunogold labelling under the electron microscope indicated Nt-Syr1 to be located primarily at the plasma membrane. Expression of the protein in leaves and to a lesser extent in flowers and stems was transiently enhanced by ABA, but not by auxin, kinetin or gibberellic acid. Expression in leaves was promoted by salt stress and wounding, but not by cold. By contrast, Nt-Syr1 levels in the root were unaffected by ABA. In the leaves, enhanced expression of Nt-Syr1 by salt stress was not observed in aba1 mutant Nicotiana, which is deficient in ABA synthesis, and in plants carrying the Arabidopsis abi1 transgene that suppresses a number of ABA-evoked responses in these plants. However, an enhanced expression in response to wounding was observed, even in the mutant backgrounds. We conclude that Nt-Syr1 expression at the plasma membrane is important for its function and is subject to control by parallel, stress-related signalling pathways, both dependent on and independent of ABA. Nt-Syr1 may be associated with additional functions, especially in the roots, that are unrelated to ABA or stress responses in the plant.

A tobacco syntaxin with a role in hormonal control of guard cell ion channels.

The plant hormone abscisic acid (ABA) regulates potassium and chloride ion channels at the plasma membrane of guard cells, leading to stomatal closure that reduces transpirational water loss from the leaf. The tobacco Nt-SYR1 gene encodes a syntaxin that is associated with the plasma membrane. Syntaxins and related SNARE proteins aid intracellular vesicle trafficking, fusion, and secretion. Disrupting Nt-Syr1 function by cleavage with Clostridium botulinum type C toxin or competition with a soluble fragment of Nt-Syr1 prevents potassium and chloride ion channel response to ABA in guard cells and implicates Nt-Syr1 in an ABA-signaling cascade.

NodS is an S-adenosyl-L-methionine-dependent methyltransferase that methylates chitooligosaccharides deacetylated at the non-reducing end.

In response to phenolic compounds exuded by the host plant, symbiotic Rhizobium bacteria produce signal molecules (Nod factors), consisting of lipochitooligosaccharides with strain-specific substitutions. In Azorhizobium caulinodans strain ORS571 these modifications are an O-arabinosyl group, an O-carbamoyl group, and an N-methyl group. Several lines of evidence indicate that the nodS gene located in the nodABCSUIJ operon is implicated in the methylation of Nod factors. Previously we have shown that NodS is an S-adenosyl-L-methionine (SAM)-binding protein, essential for the L-[3H-methyl]-methionine labelling of ORS571 Nod factors in vivo. Here, we present an in vitro assay showing that NodS from either A. caulinodans or Rhizobium species NGR234 methylates end-deacetylated chitooligosaccharides, using [3H-methyl]-SAM as a methyl donor. The enzymatic and SAM-binding activity were correlated with the nodS gene and localized within the soluble protein fraction. The A. caulinodans nodS gene was expressed in Escherichia coli and a glutathione-S-transferase-NodS fusion protein purified. This protein bound SAM and could methylate end-deacetylated chitooligosaccharides, but could not fully methylate acetylated chitooligosaccharides or unmethylated lipo-chitooligosaccharides. These data implicate that the methylation step in the biosynthesis pathway of ORS571 Nod factors occurs after deacetylation and prior to acylation of the chitooligosaccharides.

An Azorhizobium caulinodans ORS571 locus involved in lipopolysaccharide production and nodule formation on Sesbania rostrata stems and roots.

Azorhizobium caulinodans ORS571 is able to nodulate roots and stems of the tropical legume Sesbania rostrata. An ORS571 Tn5 insertion mutant, strain ORS571-X15, had a rough colony morphology, was nonmotile, and showed clumping behavior on various media. When this pleiotropic mutant was inoculated on roots or stems of the host, no nodules developed (Nod-). Compared with the wild type, strain ORS571-X15 produced lipopolysaccharides (LPS) with an altered ladder pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, suggestive of a different O-antigen structure with a lower degree of polymerization. A cosmid clone, pRG20, that fully complemented all phenotypes of ORS571-X15 was isolated. With a 6-kb EcoRI subfragment of pRG20, clumping was relieved and nodulation was almost completely restored, but the strain was still nonmotile. LPS preparations from these complemented strains resembled the wild-type LPS, although minor quantitative and qualitative differences were evident. The sequence of the locus hit by the Tn5 in ORS571-X15 (the oac locus) revealed a striking homology with the rfb locus of Salmonella typhimurium, which is involved in O-antigen biosynthesis. The Tn5 insertion position was mapped to the oac3 gene, homologous to rfbA, encoding dTDP-D-glucose synthase. Biochemical assaying showed that ORS571-X15 is indeed defective in dTDP-D-glucose synthase activity, essential for the production of particular deoxyhexoses. Therefore, it was proposed that the O antigen of the mutant strain is devoid of such sugars.