Enhanced synthesis of choline and glycine betaine in transgenic tobacco plants that overexpress phosphoethanolamine N-methyltransferase

Abstract

Choline (Cho) is the precursor of the osmoprotectant glycine betaine and is itself an essential nutrient for humans. Metabolic engineering of Cho biosynthesis in plants could therefore enhance both their resistance to osmotic stresses (drought and salinity) and their nutritional value. The key enzyme of the plant Cho-synthesis pathway is phosphoethanolamine N-methyltransferase, which catalyzes all three of the methylations required to convert phosphoethanolamine to phosphocholine. We show here that overexpressing this enzyme in transgenic tobacco increased the levels of phosphocholine by 5-fold and free Cho by 50-fold without affecting phosphatidylcholine content or growth. Moreover, the expanded Cho pool led to a 30-fold increase in synthesis of glycine betaine via an engineered glycine betaine pathway. Supplying the transgenics with the Cho precursor ethanolamine (EA) further enhanced Cho levels even though the supplied EA was extensively catabolized. These latter results establish that there is further scope for improving Cho synthesis by engineering an increased endogenous supply of EA and suggest that this could be achieved by enhancing EA synthesis and/or by suppressing its degradation.

Figure 1

Pathways of Cho and GlyBet synthesis in plants. Note that not all plants produce GlyBet. Bold arrows show the three methylation reactions that are catalyzed by PEAMT. The conversion of phospho (P-) bases to phosphatidyl (Ptd-) bases occurs via cytidine 5′-diphosphate (CDP) derivatives, which have been omitted for simplicity. MME and DME, mono- and dimethylethanolamine; Bet Ald, betaine aldehyde.

Figure 2

PEAMT expression and Cho levels in leaves of tobacco primary transformants grown in culture. (A) Immunoblot analysis of recombinant spinach PEAMT protein in leaf extracts of eight individual PEAMT⁺ transformants (tracks 1–8) and an empty-vector control (V); salinized spinach (SS) is included for comparison. Tracks contained 50 μg of protein. (B) Free Cho levels in the same plants.

Figure 3

Evidence that overexpression of PEAMT depletes the endogenous pools of EA and phosphoethanolamine in transgenic tobacco. Levels of EA, phosphoethanolamine (P-EA), and phosphomonomethylethanolamine (P-MME) were determined in leaves of transgenic line 1 expressing spinach PEAMT (PEAMT⁺) and an empty-vector control line (V). Plants were grown for 4 weeks in culture. Data are means and SE for three or four replicate plants.

Figure 4

The effect of EA supplementation on the levels of free bases and phosphobases in leaves of transgenic tobacco. Plants expressing spinach PEAMT (transgenic line 1) were grown for 4 weeks on medium with or without 5 mM EA, and the levels of free bases and phosphobases were determined in the uppermost leaves. Phosphodimethylethanolamine levels were below the detection limit (0.5 μmol g⁻¹ FW). Data are means of four replicates; asterisks denote differences between treatments that are significant at P ≤ 0.05 by ANOVA.