Expression of a Pseudomonas aeruginosa Citrate Synthase Gene in Tobacco Is Not Associated with Either Enhanced Citrate Accumulation or Efflux

Abstract

Abstract Aluminum (Al) toxicity and poor phosphorus (P) availability are factors that limit plant growth on many agricultural soils. Previous work reported that expression of a Pseudomonas aeruginosa citrate synthase gene in tobacco (Nicotiana tabacum; CSb lines) resulted in improved Al tolerance (J.M. de la Fuente, V. Ramı́rez-Rodrı́guez, J.L. Cabrera-Ponce, L. Herrera-Estrella [1997] Science 276: 1566–1568) and an enhanced ability to acquire P from alkaline soils (J. López-Bucio, O. Martı́nez de la Vega, A. Guevara-Garcı́a, L. Herrera-Estrella [2000] Nat Biotechnol 18: 450–453). These effects were attributed to the P. aeruginosa citrate synthase increasing the biosynthesis and efflux of citrate from roots. To verify these findings we: (a) characterized citrate efflux from roots of wild-type tobacco; (b) generated tobacco lines expressing the citrate synthase gene from P. aeruginosa; and (c) analyzed selected CSb lines described above. Al stimulated citrate efflux from intact roots of wild-type tobacco and root apices were found to be responsible for most of the efflux. Despite generating transgenic tobacco lines that expressed the citrate synthase protein at up to a 100-fold greater level than the previously described CSb lines, these lines did not show increased accumulation of citrate in roots or increased Al-activated efflux of citrate from roots. Selected CSb lines, similarly, failed to show differences compared with controls in either citrate accumulation or efflux. We conclude that expression of the P. aeruginosa citrate synthase gene in plants is unlikely to be a robust and easily reproducible strategy for enhancing the Al tolerance and P-nutrition of crop and pasture species.