Triterpenoid saponins are bioactive metabolites that have evolved recurrently in plants, presumably for defense. Their biosynthesis is poorly understood, as is the relationship between bioactivity and structure. Barbarea vulgaris is the only crucifer known to produce saponins. Hederagenin and oleanolic acid cellobioside make some B. vulgaris plants resistant to important insect pests, while other, susceptible plants produce different saponins. Resistance could be caused by glucosylation of the sapogenins. We identified four family 1 glycosyltransferases (UGTs) that catalyze 3-O-glucosylation of the sapogenins oleanolic acid and hederagenin. Among these, UGT73C10 and UGT73C11 show highest activity, substrate specificity and regiospecificity, and are under positive selection, while UGT73C12 and UGT73C13 show lower substrate specificity and regiospecificity and are under purifying selection. The expression of UGT73C10 and UGT73C11 in different B. vulgaris organs correlates with saponin abundance. Monoglucosylated hederagenin and oleanolic acid were produced in vitro and tested for effects on P. nemorum. 3-Ob-D-Glc hederagenin strongly deterred feeding, while 3-O-b-D-Glc oleanolic acid only had a minor effect, showing that hydroxylation of C23 is important for resistance to this herbivore. The closest homolog in Arabidopsis thaliana, UGT73C5, only showed weak activity toward sapogenins. This indicates that UGT73C10 and UGT73C11 have neofunctionalized to specifically glucosylate sapogenins at the C3 position and demonstrates that C3 monoglucosylation activates resistance. As the UGTs from both the resistant and susceptible types of B. vulgaris glucosylate sapogenins and are not located in the known quantitative trait loci for resistance, the difference between the susceptible and resistant plant types is determined at an earlier stage in saponin biosynthesis.

To clarify the role of mitochondrial small heat-shock protein (MT-sHSP) in the heat-shock response, we introduced the tomato (Lycopersicon esculentum) MT-sHSP gene under the control of the 35S promoter into tobacco (Nicotiana tabacum), and examined the thermotolerance of the transformed plants. Irrespective of the orientation, sense or antisense, of the gene, the transgenic plants exhibited a normal morphology and growth rate in the vegetative growth stage. When 4-weekold seedlings were exposed to sudden heat stress, the sense plants which overexpress the MT-sHSP gene exhibited thermotolerance, whereas the antisense plants in which the expression of the gene is suppressed exhibited susceptibility.

We cloned a cDNA encoding Hordeum vulgare Proline Transporter (HvProT) from salt-stressed barley roots by differential display. HvProT was 2,161 bp long and had an open reading frame encoding 450 amino acids. The deduced amino acid sequence of HvProT was similar to those of proline transporter proteins of rice (65.7%), Arabidopsis (57.7%) and tomato (42.0%). Northern blot analysis showed that the transcript level of HvProT was induced in roots at 30 min after 200 mM NaCl treatment and its peak was observed at 3 h. However, the transcript level was very low in leaves and did not increase by salt stress. The expression level of Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), encoding a key enzyme of proline synthesis, was induced later than HvProT by salt stress. A transport assay using a yeast with mutation in proline uptake revealed that HvProT was a transporter with high affinity for L-proline (K(m) = 25 microM). HvProT was found to be a unique transporter with high affinity for L-proline. Since its transport activity was dependent on the pH gradient, HvProT was suggested to be a H(+)/amino acid symporter. In situ hybridization analysis showed that the HvProT mRNA was strongly expressed in root cap cells under salt stress. HvProT might play an important role in the transport of proline to root tip region urgently upon salt stress.

The subcellular localization of plant farnesyl diphosphate synthase (FPPS) was examined. Immunocytochemical staining using anti-FPPS1 antibody followed by electron microscopy showed that FPPS1 was localized to chloroplasts of rice mesophyll cells. Subcellular fractions from wheat leaves were examined by immunoblot analysis. FPPS was detected in the chloroplast fraction in wheat, and was protected from proteolysis following trypsin treatment of chloroplasts. FPPS was also detected in the chloroplast fraction of a dicot plant, tobacco.

Mx8 is a generalized transducing phage that infects Myxococcus xanthus cells. This phage is lysogenized inM. xanthus cells by the integration of its DNA into the host chromosome through site-specific recombination. Here, we characterize the mechanism of Mx8 integration into the M. xanthus chromosome. The Mx8 attachment site, attP, the M. xanthus chromosome attachment site, attB, and two phage-host junctions, attL and attR, were cloned and sequenced. Sequence alignments of attP, attB, attL, and attR sites revealed a 29-bp segment that is absolutely conserved in all four sequences. The intP gene of Mx8 was found to encode a basic protein that has 533 amino acids and that carries two domains conserved in site-specific recombinases of the integrase family. Surprisingly, the attP site was located within the coding sequence of the intP gene. Hence, the integration of Mx8 into the M. xanthus chromosome results in the conversion of the intP gene to a new gene designated intR. As a result of this conversion, the 112-residue C-terminal sequence of the intP protein is replaced with a 13-residue sequence. A 3-base deletion within the C-terminal region had no effect on Mx8 integration into the chromosome, while a frameshift mutation with the addition of 1 base at the same site blocked integration activity. This result indicates that the C-terminal region is required for the enzymatic function of the intP product.Myxococcus xanthus is a unique gram-negative bacterium living in soil. M. xanthus cells can undergo multicellular development involving cell-to-cell interactions (for a review, see reference 8). Upon nutritional starvation on a solid surface, cells aggregate to form mounds called fruiting bodies within which rod-shaped cells are converted into spherical or ovoid myxospores.Several bacteriophages that infect M. xanthus cells are known (17). Myxophage Mx8 is a generalized transducing phage of M. xanthus (22). Purified phage particles have 56-kb linear double-stranded DNA molecules with an average terminal redundancy of 4.3 kb (31). Restriction analyses showed that Mx8 phage DNA is circularly permuted (see Fig. 1A). This phage can be lysogenized in M. xanthus cells by integrating its DNA into the host chromosome through site-specific recombination between the attP site on the phage DNA and the attB site on the host chromosome (4, 25). This recombination system has been used to introduce recombinant plasmids into the M. xanthus chromosome, since various plasmids containing a fragment of Mx8 DNA have been shown to stably integrate into the chromosomal attB site (15,29,31). In spite of the effectiveness and wide utilization of the Mx8 attP-mediated integration of plasmids, the integration mechanism is not well understood at present.To reveal the mechanism of Mx8 site-specific recombination in M. xanthus, we analyzed the intP-attP region of Mx8 phage.Comparison of attP, attB, attL, and attR sequences revealed a 29-bp segment that is absolutely conserved. The Mx8 intP gene was shown to encode a basic protein with 533 amino a...