Previous work with model transgenic plants has demonstrated that cellular accumulation of mannitol can alleviate abiotic stress. Here, we show that ectopic expression of the mtlD gene for the biosynthesis of mannitol in wheat improves tolerance to water stress and salinity. Wheat (Triticum aestivum L. cv Bobwhite) was transformed with the mtlD gene of Escherichia coli. Tolerance to water stress and salinity was evaluated using calli and T 2 plants transformed with (ϩmtlD) or without (ϪmtlD) mtlD. Calli were exposed to Ϫ1.0 MPa of polyethylene glycol 8,000 or 100 mm NaCl. T 2 plants were stressed by withholding water or by adding 150 mm NaCl to the nutrient medium. Fresh weight of ϪmtlD calli was reduced by 40% in the presence of polyethylene glycol and 37% under NaCl stress. Growth of ϩmtlD calli was not affected by stress. In ϪmtlD plants, fresh weight, dry weight, plant height, and flag leaf length were reduced by 70%, 56%, 40%, and 45% compared with 40%, 8%, 18%, and 29%, respectively, in ϩmtlD plants. Salt stress reduced shoot fresh weight, dry weight, plant height, and flag leaf length by 77%, 73%, 25%, and 36% in ϪmtlD plants, respectively, compared with 50%, 30%, 12%, and 20% in ϩmtlD plants. However, the amount of mannitol accumulated in the callus and mature fifth leaf (1.7-3.7 mol g Ϫ1 fresh weight in the callus and 0.6-2.0 mol g Ϫ1 fresh weight in the leaf) was too small to protect against stress through osmotic adjustment. We conclude that the improved growth performance of mannitol-accumulating calli and mature leaves was due to other stress-protective functions of mannitol, although this study cannot rule out possible osmotic effects in growing regions of the plant.Water stress and salinity are major abiotic factors that limit crop productivity in drought-prone areas. One way of increasing productivity in stressful environments is to breed crops that are more tolerant to stress. However, success in breeding for tolerance has been limited because (a) tolerance to stress is controlled by many genes, and their simultaneous selection is difficult (Richards, 1996;Yeo, 1998;Flowers et al., 2000); (b) tremendous effort is required to eliminate undesirable genes that are also incorporated during breeding (Richards, 1996); and (c) there is a lack of efficient selection procedures particularly under field conditions (Ribaut et al., 1997). Genetic engineering offers an alternative approach for developing tolerant crops. Unlike classical breeding, genetic engineering is a faster and more precise means of achieving improved tolerance (Cushman and Bohnert, 2000) because it avoids the transfer of unwanted chromosomal regions. Moreover, through genetic engineering, multiple genes can be assembled and simultaneously introduced to the crop of interest. There are many functional targets for engineering tolerance to water stress and salinity, one of them being accumulation of osmoprotectants (Rathinasabapathi, 2000).The osmolyte mannitol is normally synthesized in numerous plant species, but not in wheat (Triticum aes...

Fusarium head blight (FHB), primarily caused by Fusarium graminearum Schw., is a destructive disease of wheat (Triticum aestivum L.). Although several genes related to FHB resistance have been reported, global analysis of gene expression in response to FHB infection remains to be explored. The expression patterns of transcriptomes from wheat spikes of FHB-resistant cultivar Ning 7840 and susceptible cultivar Clark were monitored during a period of 72 h after inoculation (hai) with F. graminearum. Microarray analysis, coupled with suppression subtractive hybridization technique, identified 44 significantly differentially expressed genes between cv. Ning 7840 and cv. Clark. More differentially expressed genes were identified from susceptible libraries than from resistance libraries. The up-regulation of defense-related genes in Ning 7840 relative to cultivar Clark occurred during early fungal stress (3-12 hai). Three genes, with unknown function that were up-regulated in cv. Ning 7840 at most time points investigated, might play an important role in enhancing FHB resistance.

Four major genes in wheat (Triticum aestivum L.), with the dominant alleles designated Vrn-A1, Vrn-B1, Vrn-D1, and Vrn4, are known to have large effects on the vernalization response, but the effects on cold hardiness are ambiguous. Near-isogenic experimental lines (NILs) in a Triple Dirk (TD) genetic background with different vernalization alleles were evaluated for cold hardiness. Although TD is homozygous dominant for Vrn-A1 (formerly Vrn1) and Vrn-B1 (formerly Vrn2), four of the lines are each homozygous dominant for a different vernalization gene, and one line is homozygous recessive for all four vernalization genes. Following establishment, the plants were initially acclimated for 6 weeks in a growth chamber and then stressed in a low temperature freezer from which they were removed over a range of temperatures as the chamber temperature was lowered 1.3 degrees C h(-1). Temperatures resulting in no regrowth from 50% of the plants (LT(50)) were determined by estimating the inflection point of the sigmoidal response curve by nonlinear regression. The LT(50) values were -6.7 degrees C for cv. TD, -6.6 degrees C for the Vrn-A1 and Vrn4 lines, -8.1 degrees C for the Vrn-D1 (formerly Vrn3) line, -9.4 degrees C for the Vrn-B1 line, and -11.7 degrees C for the homozygous recessive winter line. The LT(50) of the true winter line was significantly lower than those of all the other lines. Significant differences were also observed between some, but not all, of the lines possessing dominant vernalization alleles. The presence of dominant vernalization alleles at one of the four loci studied significantly reduced cold hardiness following acclimation.

Coronamic acid (CMA; 2-ethyl-1-aminocyclopropane 1-carboxylic acid) is an intermediate in the biosynthesis of coronatine (COR), a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. Tn5 mutagenesis and substrate feeding studies were previously used to characterize regions of the COR biosynthetic gene cluster required for synthesis of coronafacic acid and CMA, which are the only two characterized intermediates in the COR biosynthetic pathway. In the present study, additional Tn5 insertions were generated to more precisely define the region required for CMA biosynthesis. A new analytical method for CMA detection which involves derivatization with phenylisothiocyanate and detection by high-performance liquid chromatography (HPLC) was developed. This method was used to analyze and quantify the production of CMA by selected derivatives of P. syringae pv. glycinea which contained mutagenized or cloned regions from the CMA biosynthetic region. pMU2, a clone containing a 6.45-kb insert from the CMA region, genetically complemented mutants which required CMA for COR production. When pMU2 was introduced into P. syringae pv. glycinea 18a/90 (a strain which does not synthesize COR or its intermediates), CMA was not produced, indicating that pMU2 does not contain the complete CMA biosynthetic gene cluster. However, when two plasmid constructs designated pMU234 (12.5 kb) and pKTX30 (3.0 kb) were cointroduced into 18a/90, CMA was detected in culture supernatants by thin-layer chromatography and HPLC. The biological activity of the CMA produced by P. syringae pv. glycinea 18a/90 derivatives was demonstrated by the production of COR in cosynthesis experiments in which 18a/90 transconjugants were cocultivated with CMA-requiring mutants of P. syringae pv. glycinea PG4180. CMA production was also obtained when pMU234 and pKTX30 were cointroduced into P. syringae pv. syringae Bi; however, these two constructs did not enable Escherichia coli K-12 to synthesize CMA. The production of CMA in P. syringae strains which lack the COR biosynthetic gene cluster indicates that CMA production can occur independently of coronafacic acid biosynthesis and raises interesting questions regarding the evolutionary origin of the COR biosynthetic pathway. Coronatine (COR) is a chlorosis-inducing non-host-specific phytotoxin produced by several Pseudomonas syringae patho