Glutamate decarboxylase (GAD; EC 4.1.1.15) catalyzes the irreversible decarboxylation of glutamate to produce γ-aminobutyric acid (GABA); a ubiquitous non-protein amino acid involved in the regulation of several aspects of plant metabolism and physiology. To study the function of GAD and GABA in maize, we have; 1) introduced native and deregulated forms of AtGAD1 into maize with the intent of increasing the synthesis of GABA and 2) introduced constructs into maize designed to suppress the activity of several GABA shunt, GABA transport and GABA pathway genes. Maize plants expressing the deregulated AtGAD1 exhibit a severe chlorosis and retarded growth phenotype and have high levels of GABA, and Ca⁺⁺/CaM-independent GAD activity. Plants expressing the suppression constructs for GABA biosynthetic and transport pathway genes had no observable phenotype whereas a knockout of GABA catabolic pathway genes led to growth and developmental defects under standard growth conditions. The implications of this study to our understanding of the action and function of GABA and GAD in crops are discussed.

In an attempt to increase source capacity, transgenic corn was generated by expressing a truncated maize sucrose phosphate synthase (ZmSPSΔ482) under two leaf mesophyll cellspecific promoters (CAB and PPDK). The endogenous and truncated SPS proteins from transgenic leaf extracts were distinguishable by protein immunoblot analysis. The expression of transgenic SPS protein across events varied from very low to very high and included several cosuppressed events. SPS activity showed a diurnal pattern in both transgenic and wild-type maize leaves. In greenhouse experiments, transgenic maize had higher leaf sucrose and lower leaf starch, suggesting a shift in carbon partitioning from starch to sucrose. Conversely, cosuppressed events had lower leaf sucrose and higher leaf starch. A field test was performed to compare sucrose and starch in positive and negative isolines of hybrid maize CAB and PPDK ZmSPSΔ482 events. In the field, many positive isolines had higher levels of both leaf sucrose and starch than the negative isolines. This suggests that in the field, with higher light intensity the shift in carbon partitioning from starch to sucrose, observed under greenhouse conditions did not occur. This in turn suggests that the environment affects the phenotype of the transgenics and that in the field, there was an overall increase in carbon assimilation. Six events from each construct were tested in a pilot multi-density yield trial but overall, no effect on yield was observed. Therefore, although the transgenic plants had more source capacity, this did not translate into higher seed yield.

Glyphosate (N-phosphonomethylglycine) is a broad-spectrum, foliar-applied herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in plants. EPSPS catalyzes a crucial step in the shikimate pathway for the biosynthesis of folates and aromatic amino acids in plants. A variety of glyphosate-tolerant EPSPS enzymes have been reported. Some of these have been introduced into crops using biotechnology to produce glyphosate-tolerant crops. Glyphosate tolerance in crops permits the use of glyphosate to control weeds while maintaining crop yield. We endeavored to optimize the maize EPSPS enzyme with improvements in both enzymatic activity and reduction in sensitivity to glyphosate to improve the potential for herbicide tolerance in crops. Here, we have improved the glyphosate tolerance of maize EPSPS with the potential of providing an herbicide tolerance trait by utilizing enzyme optimization with in vitro and in planta screening. Overexpressing some of these EPSPS variants into maize have resulted in maize plants with robust vegetative glyphosate tolerance.

Optimized triketone dioxygenase (TDO) variants with enhanced temperature stability parameters were engineered to enable robust triketone tolerance in transgenic cotton and soybean crops. This herbicide tolerance trait, which can metabolize triketone herbicides such as mesotrione and tembotrione, could be useful for weed management systems and provide additional tools for farmers to control weeds. TDO has a low melting point (~39°C–40°C). We designed an optimization scheme using a hypothesis-based rational design to improve the temperature stability of TDO. Temperature stabilization resulted in enzymes with K_cat values less than half of wild-type TDO. The best variant TDO had a K_cat of 1.2 min⁻¹ compared to wild-type TDO, which had a K_cat of 2.7 min⁻¹. However K_m values did not change much due to temperature stabilization. Recovery of the K_cat without losing heat stability was the focus of additional optimization. Multiple variants were found that had better heat stability in vitro and efficacies against mesotrione equaling the wild-type (WT) TDO in greenhouse and field tests.