Synthetic glycolate metabolism pathways stimulate crop growth and productivity in the field

INTRODUCTION Meeting food demands for the growing global human population requires improving crop productivity, and large gains are possible through enhancing photosynthetic efficiency. Photosynthesis requires the carboxylation of ribulose-1,5-bisphosphate (RuBP) by ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO), but photorespiration occurs in most plants such as soybean, rice, and wheat (known as C 3 crops) when RuBisCO oxygenates RuBP instead, requiring costly processing of toxic byproducts such as glycolate. Photorespiration can reduce C 3 crop photosynthetic efficiency by 20 to 50%. Although various strategies exist for lowering the costs of photorespiration, chamber- and greenhouse-grown plants with altered photorespiratory pathways within the chloroplast have shown promising results, including increased photosynthetic rates and plant size. RATIONALE To determine if alternative photorespiratory pathways could effectively improve C 3 field crop productivity, we tested the performance of three alternative photorespiratory pathways in field-grown tobacco. One pathway used five genes from the Escherichia coli glycolate oxidation pathway; a second pathway used glycolate oxidase and malate synthase from plants and catalase from E. coli ; and the third pathway used plant malate synthase and a green algal glycolate dehydrogenase. All enzymes in the alternative pathway designs were directed to the chloroplast. RNA interference (RNAi) was also used to down-regulate a native chloroplast glycolate transporter in the photorespiratory pathway, thereby limiting metabolite flux through the native pathway. The three pathways were introduced with and without the transporter RNAi construct into tobacco, which is an ideal model field crop because it is easily transformed, has a short life cycle, produces large quantities of seed, and develops a robust canopy similar to that of other field crops. RESULTS Using a synthetic biology approach to vary promoter gene combinations, we generated a total of 17 construct designs of the three pathways with and without the transporter RNAi construct. Initial screens for photoprotection by alternative pathway function under high–photorespiratory stress conditions identified three to five independent transformants of each design for further analysis. Gene and protein expression analyses confirmed expression of the introduced genes and suppression of the native transporter in RNAi plants. In greenhouse screens, pathway 1 increased biomass by nearly 13%. Pathway 2 showed no benefit compared to wild type. Introduction of pathway 3 increased biomass by 18% without RNAi and 24% with RNAi, which were consistent with changes in photorespiratory metabolism and higher photosynthetic rates. Ultimately, field testing across two different growing seasons showed >25% increase in biomass of pathway 3 plants compared to wild type, and with RNAi productivity increased by >40%. In addition, this pathway increased the light-use efficiency of photosynthesis by 17% in the field. CONCLUSION Engineering more efficient photorespiratory pathways into tobacco while inhibiting the native pathway markedly increased both photosynthetic efficiency and vegetative biomass. We are optimistic that similar gains may be achieved and translated into increased yield in C 3 grain crops because photorespiration is common to all C 3 plants and higher photosynthetic rates under elevated CO 2 , which suppresses photorespiration and increases harvestable yield in C 3 crops.