Broad 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor Herbicide Tolerance in Soybean with an Optimized Enzyme and Expression Cassette  

Abstract

With an optimized expression cassette consisting of the soybean (Glycine max) native promoter modified for enhanced expression driving a chimeric gene coding for the soybean native amino-terminal 86 amino acids fused to an insensitive shuffled variant of maize (Zea mays) 4-hydroxyphenylpyruvate dioxygenase (HPPD), we achieved field tolerance in transgenic soybean plants to the HPPD-inhibiting herbicides mesotrione, isoxaflutole, and tembotrione. Directed evolution of maize HPPD was accomplished by progressively incorporating amino acids from naturally occurring diversity and novel substitutions identified by saturation mutagenesis, combined at random through shuffling. Localization of heterologously expressed HPPD mimicked that of the native enzyme, which was shown to be dually targeted to chloroplasts and the cytosol. Analysis of the native soybean HPPD gene revealed two transcription start sites, leading to transcripts encoding two HPPD polypeptides. The N-terminal region of the longer encoded peptide directs proteins to the chloroplast, while the short form remains in the cytosol. In contrast, maize HPPD was found almost exclusively in chloroplasts. Evolved HPPD enzymes showed insensitivity to five inhibitor herbicides. In 2013 field trials, transgenic soybean events made with optimized promoter and HPPD variant expression cassettes were tested with three herbicides and showed tolerance to four times the labeled rates of mesotrione and isoxaflutole and two times the labeled rates of tembotrione.

Physiological and Molecular Characterization of Hydroxyphenylpyruvate Dioxygenase (HPPD)-inhibitor Resistance in Palmer Amaranth (Amaranthus palmeri S.Wats.)

“…Tobacco, a dicot species, is highly sensitive to mesotrione, however, when transformed with a HPPD gene from wheat, showed tolerance to this herbicide (Hawkes et al, 2001). Transgenic soybeans tolerant to mesotrione, tembotrione, and isoxaflutole have been developed with an herbicide-insensitive maize HPPD to increase the selectivity and spectrum of weed control (Siehl et al, 2014). Mesotrione and other HPPD-inhibitors are important in controlling several ALS- and PS II-inhibitor resistant weed biotypes (Sutton et al, 2002).…”

Free energy calculations elucidate substrate binding, gating mechanism, and tolerance‐promoting mutations in herbicide target 4‐hydroxyphenylpyruvate dioxygenase

“…Herbicide tolerance is typically assessed by comparing the inhibition of wild type and mutant HPPD proteins by different inhibitors. A range of different mutations has been proposed for HPPD proteins in mousear cress, rice, and soybean . Recently, Siehl et al presented an approach to develop transgenic soybean plants tolerant to a variety of HPPD inhibitors by directed evolution of maize HPPD .…”

“…These mutations did not involve any residues reported to be related to catalytic function . The authors concluded that the improved herbicide tolerance occurred “through the accumulated effect of many small changes outside the active site that influence the catalytic properties of the enzyme in a manner that is not obvious upon inspection of the structure.” Hence, a detailed understanding of the molecular phenomena promoting herbicide tolerance and especially the effect of mutations outside of the active site is lacking to date.…”

Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds

“…Given this scenario, reduced catabolism of homogentisate in MO12 cells would therefore increase the effective levels of homogentisate for translocation into plastids for tocochromanol production. However, it was recently reported that homogentisate can be synthesized in the plastids of soybean and likely other legumes as well (Siehl et al, 2014). This would indicate that leguminous plants can utilize plastidic homogentisate directly for secondary metabolism without necessitating transport into the plastids, as is the case in other plants.…”