Characterizing the Response of gdhA Transformed Tobacco to Glufosinate

Author

Scott Nolte, Southern Illinois University CarbondaleFollow

Date of Award

12-2009

Degree Name

Doctor of Philosophy

Department

Plant Biology

First Advisor

Young, Bryan

Second Advisor

Lightfoot, David

Abstract

The gene gdhA from Escherichia coli, that encodes a NADPH-dependent glutamate dehydrogenase (GDH), directs a novel pathway in transgenic plants that potentially allows an increase in ammonium assimilation. Glufosinate leads to plant death by the irreversible inhibition of glutamate synthetase (GS) leading to a disruption of subsequent GS-related processes resulting in elevated ammonium and disruption of photorespiration. Therefore, it was speculated that the gdhA-transformed plants may exhibit a novel mechanism of resistance to glufosinate by altered activity of the GDH pathway and subsequently related processes. Studies were conducted in the greenhouse to evaluate 1) whole plant tolerance to glufosinate, 2) changes in absorption, translocation and metabolism of glufosinate, and 3) metabolic fingerprint changes in response to glufosinate treatment in tobacco plants containing the gdhA gene. Whole plant tolerance experiments showed that tobacco transformed with the gdhA gene expressed up to six fold increased resistance (GR₅₀) to glufosinate compared with the non-gdhA control line. GDH enzyme activity among gdhA-transformed tobacco lines was highly correlated (r2 = 0.9903) with the amount of herbicide resistance. Thus, use of the E. coli gdhA gene in plant transformations can provide an additional mechanism for resistance to glufosinate. Foliar absorption and translocation of 14C from glufosinate was not altered to any large extent in gdhA-transformed plants which suggests these factors cannot fully explain the mechanism for whole-plant resistance to glufosinate. However, the metabolic fingerprint resulting from glufosinate treatment was significantly altered in gdhA tobacco. It was also shown that metabolic perturbation induced by glufosinate was lower in the high GDH activity tobacco line, +gdhA 9, than in the non-gdhA control tobacco line as evidenced by the reduced number of altered peaks recorded in leaves of these two tobacco lines. Thus, gdhA-transformed tobacco plants with low and high expression of GDH activity, exhibited greater overall stability of metabolism following the application of glufosinate, than recorded in non-gdhA control plants. This greater metabolic stability during GS inhibition was likely due to the amelioration of amino acid production through the increased activity of GDH. Therefore, the hypothesized mechanism of increased resistance to glufosinate in gdhA-transformed tobacco lines is by maintenance of amino acid production and maintenance of photorespiratory activity.