ABSTRACT
Engineering plant vegetative tissue to accumulate triacylglycerols (TAG, e.g., oil) can increase the amount of oil harvested per acre to levels that exceed current oilseed crops. Engineered Nicotiana tabacum (tobacco) lines that accumulate 15% to 30% oil of leaf dry weight resulted in starkly different metabolic phenotypes. In-depth analysis of the leaf lipid accumulation and 14CO2 metabolic tracing mechanistically described metabolic adaptations to the leaf oil engineering. An oil-for-membrane lipid tradeoff in the 15% oil line (referred to as HO) was surprisingly not further exacerbated when lipid production was enhanced to 30% (LEC2 line). The HO line exhibited a futile cycle that limited TAG yield through exchange with starch, altered carbon flux into various metabolite pools and end products, and suggested overlapping pathways of the glyoxylate cycle and photorespiration that limited CO2 assimilation by 50%. In contrast, inclusion of the LEC2 transcription factor in tobacco improved TAG stability, alleviated the TAG-to-starch futile cycle, and recovered CO2 assimilation and plant growth comparable to wild type but with much higher lipid levels in the leaves. Thus, the unstable production of storage reserves and futile cycling limit vegetative oil engineering approaches. The capacity to overcome futile cycles and maintain enhanced stable TAG levels in LEC2 demonstrated the importance of considering unanticipated metabolic adaptations while engineering vegetative oil crops.
