The endogenous choline supply limits glycine betaine synthesis in transgenic tobacco expressing choline monooxygenase

Abstract

Summary: Certain plants produce glycine betaine (GlyBet) in the chloroplast by a two‐step oxidation of choline. Introducing GlyBet accumulation into plants that lack it is a well‐established target for metabolic engineering because GlyBet can lessen damage from osmotic stress. The first step in GlyBet synthesis is catalyzed by choline monooxygenase (CMO), a stromal enzyme with a Rieske‐type [2Fe‐2S] center. The absence of CMO is the primary constraint on GlyBet production in GlyBet‐deficient plants such as tobacco, but the endogenous choline supply is also potentially problematic. To investigate this, we constructed transgenic tobacco plants that constitutively express a spinach CMO cDNA. The CMO protein was correctly compartmented in chloroplasts and was enzymatically active, showing that its [2Fe‐2S] cluster had been inserted. Salinization increased CMO protein levels, apparently via a post‐transcriptional mechanism, to as high as 10% of that in salinized spinach. However, the GlyBet contents of CMO+ plants were very low (0.02– 0.05 μmol g–1 fresh weight) in both unstressed and salinized conditions. Experiments with [14 C]GlyBet demonstrated that this was not due to GlyBet catabolism. When CMO+ plants were supplied in culture with 5 mM choline or phosphocholine, their choline and GlyBet levels increased by at least 30‐fold. The choline precursors mono‐ and dimethylethanolamine also enhanced choline and GlyBet levels but ethanolamine did not, pointing to a major constraint on flux to choline at the first methylation step in its synthesis. The extractable activity of the enzyme mediating this step in tobacco was only 3% that of spinach. We conclude that in GlyBet‐deficient plants engineered with choline‐oxidizing genes, the size of the free choline pool and the metabolic flux to choline need to be increased to attain GlyBet levels as high as those in natural accumulators.