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.
Highlights
To functionally corroborate the localization of maize hydroxyphenylpyruvate dioxygenase (HPPD), a binary plant transformation vector was constructed in which the portion of the maize HPPD gene coding for the N-terminal 50 amino acids was fused to the gene coding for Discosoma spp
Transformation of guard cells with vectors encoding either Rubisco activase chloroplast-targeting peptide (CTP)-Discosoma spp. RED FLUORESCENT PROTEIN2 (DsRed2) or the N-terminal 50 amino acids of maize HPPD fused to DsRed2 clearly resulted in plastid targeting of the DsRed2 reporter, whereas untargeted cycle 3 GFP (C3GFP) showed no overlap with the DsRed2 signal (Supplemental Fig. S2)
In addition to a steadily increasing inventory of glyphosate-resistant weeds, there are documented cases of weeds, such as Amaranthus spp., that are resistant to multiple herbicides, including acetolactate synthase inhibitors, atrazine, protoporphyrinogen oxidase inhibitors, HPPD inhibitors, and glyphosate (Heap, 2014)
Summary
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. 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. While favored for its efficacy, economy, and convenience, the onset of glyphosate-resistant weeds, numbering 29 species (Heap, 2014), signals a need for new herbicide and trait systems as effective as glyphosate (Green and Castle, 2010; Duke, 2012). Two transgenic soybean crop products, one for tolerance to isoxaflutole (APHIS, 2009) and the other to mesotrione
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