Development of THA mutant line having markedly increased threonine in cured leaves
Threonine (Thr), an amino acid, is known to be related to tobacco smoking flavour and aroma. Obtaining Thr-rich cured tobacco leaves is difficult because Thr contents in fresh tobacco leaves are decreased drastically during curing processes. To increase Thr contents in plants, the modification of aspartate kinase (AK), the key enzyme of Thr biosynthesis, is a major approach, but growth inhibition occurs in the AK modified mutant. As another approach, we specifically emphasized the inactivation of threonine aldorase (THA), which is a key enzyme regulating Thr degradation. It is known for its high expression during the senescence stage rather than the growth stage.
This study investigated how the knockout mutation of the THA gene affects Thr accumulation in cured tobacco leaves and tobacco growth. First, each mutant tobacco plant having nonsense mutation of NtTHA1-S encoded in the S-genome derived from N. sylvestris, and NtTHA1-T encoded in the T-genome derived from N. tomentosiformis was screened from the ethyl ethanesulfonate-mutagenized tobacco library (var. Tsukuba 1: flue-cured tobacco). Subsequently, double mutant lines (sstt) and control lines (SSTT) were obtained by crossing between single mutants. Double mutant lines and control lines were cultivated in the field. Then leaves (leaf position) were harvested 50 days after topping. The Thr content in the harvested fresh leaves showed no clear difference between the double mutant and the control. In contrast, after flue-curing, the Thr content in double mutant leaves was higher than that of the control. Furthermore, agronomic performance of double mutant (sstt) was not different from that of control. Double mutant line (sstt) backcrossed with Burley (var. TN 90) was also evaluated. The result was identical to that found for the flue-cured tobacco mutant. In conclusion, THA mutation is a good measure to increase Thr levels in cured tobacco leaves without affecting tobacco growth or yield.