Tolerance of mannitol-accumulating transgenic wheat to water stress and salinity

Abstract

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 (+mtlD) or without (-mtlD) mtlD. Calli were exposed to -1.0 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 -mtlD calli was reduced by 40% in the presence of polyethylene glycol and 37% under NaCl stress. Growth of +mtlD calli was not affected by stress. In -mtlD 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 +mtlD plants. Salt stress reduced shoot fresh weight, dry weight, plant height, and flag leaf length by 77%, 73%, 25%, and 36% in -mtlD plants, respectively, compared with 50%, 30%, 12%, and 20% in +mtlD plants. However, the amount of mannitol accumulated in the callus and mature fifth leaf (1.7-3.7 micromol g(-1) fresh weight in the callus and 0.6-2.0 micromol g(-1) 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.

Figure 1

Plasmids used for wheat transformation. Plasmid pAHC20 contains only the selectable marker bar. Plasmid pTA2 contains bar and the E. coli mtlD gene for biosynthesis of mannitol-1-phosphate. Both genes were under the control of the maize (Zea mays) ubi-1 promoter. Calli and plants transformed with pTA2 were used as mannitol-accumulating lines (+mtlD), and those transformed with pAHC20 served as negative controls (−mtlD).

Figure 2

Effect of osmotic stress on the growth of transgenic wheat calli. The mannitol-accumulating callus line C2-20 (+mtlD) and the nonaccumulating line C1-11 (−mtlD) were grown in Murashige and Skoog medium containing PEG 8,000 (−1.0 MPa) or 100 mm NaCl for 60 d.

Figure 3

Effect of water stress and salinity on the growth of +mtlD and −mtlD plants. The mannitol-accumulating transgenic wheat line P2-19-1 (+mtlD) and the nonaccumulating P1-13-1 (−mtlD) were stressed by withholding water (A) or by supplementing the nutrient solution with 150 mm NaCl (B) for 30 d. Pictures were taken 30 d after the imposition of water stress and 20 d after NaCl stress. In the absence of stress, −mtlD and +mtlD plants were similar in size; thus, for unstressed controls, only the −mtlD plants are shown.

Figure 4

Phenotypes observed in transgenic wheat plants. Lines P2-16-1 and P2-19-1 were transformed with plasmid pTA2 for accumulation of mannitol in the cytosol (+mtlD). Line P1-13-1 was transformed with pAHC20 (−mtlD) and did not accumulate mannitol. Most +mtlD plants were short and sterile and had twisted leaves and heads similar to P2-16-1. In addition, the sterile plants had high mannitol (more than 1.5 μmol g⁻¹ fresh weight) and low Suc content (less than 2 μmol g⁻¹ fresh weight). In the fertile +mtlD plants, mannitol content ranged from 0.4 to 0.7 μmol g⁻¹ fresh weight.