Genetic Engineering of Glycinebetaine Production toward Enhancing Stress Tolerance in Plants: Metabolic Limitations

Abstract

Glycinebetaine (betaine) affords osmoprotection in bacteria, plants and animals, and protects cell components against harsh conditions in vitro. This and a compelling body of other evidence have encouraged the engineering of betaine production in plants lacking it. We have installed the metabolic step for oxidation of choline, a ubiquitous substance, to betaine in three diverse species, Arabidopsis, Brassica napus, and tobacco (Nicotiana tabacum), by constitutive expression of a bacterial choline oxidase gene. The highest levels of betaine in independent transgenics were 18.6, 12.8, and 13 mol g ؊1 dry weight, respectively, values 10-to 20-fold lower than the levels found in natural betaine producers. However, choline-fed transgenic plants synthesized substantially more betaine. Increasing the choline supplementation further enhanced betaine synthesis, up to 613 mol g ؊1 dry weight in Arabidopsis, 250 mol g ؊1 dry weight in B. napus, and 80 mol g ؊1 dry weight in tobacco. These studies demonstrate the need to enhance the endogenous choline supply to support accumulation of physiologically relevant amounts of betaine. A moderate stress tolerance was noted in some but not all betaine-producing transgenic lines based on relative shoot growth. Furthermore, the responses to stresses such as salinity, drought, and freezing were variable among the three species.

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

“…It should be noted that this constraint is missing from plants engineered with a cytosolic Cho 3 GlyBet pathway by using bacterial Cho-oxidizing enzymes (3,5,19), so that overexpressing PEAMT in such plants could lead to higher GlyBet levels than those we attained. Because the GlyBet levels we achieved are comparable to those already demonstrated by several groups to enhance stress resistance in tobacco and other plants (5,25,31), we did not reduplicate this work with our transgenics.…”

“…However, the levels of GlyBet thus far attained by engineering are low, and the increments in stress tolerance are commensurately small (4). It has been shown that the low levels of GlyBet in the engineered plants are due in large part to an inadequate capacity for Cho synthesis (4)(5)(6). This constraint on GlyBet synthesis provides one reason to enhance Cho biosynthesis in plants.…”

Glycinebetaine improves salt tolerance in vinal (Prosopis ruscifolia Griesbach) seedlings

“…Exogenous application of GB to rice plants growing under salt stress resulted in reduced Na + accumulation and the maintenance of K + concentrations in the shoot (Lutts, 2000). In a number of studies carried out with transgenic plants engineered to express higher levels of GB, the levels of accumulated GB were rarely more than 1 µmol g -1 FW (Holmström et al, 2000;Huang et al, 2000). This is far too low for conventional osmorregulation, even assuming that all of these solutes are located in the cytosol.…”

Regulation of phosphatidylcholine biosynthesis under salt stress involves choline kinases in Arabidopsis thaliana

“…Much effort has been undertaken to improve stress resistance in model species, such as tobacco and Arabidopsis, that do not possess the Cho monooxygenase nor the betaine aldehyde dehydrogenase enzymes necessary for GB biosynthesis (see [14] for a review). In Cho monooxygenase transgenic tobacco, significant accumulation of GB occurs only after exogenous Cho supply or by engineering plants to overproduce Cho, and it appears that Cho biosynthesis is a major limiting factor in GB synthesis [13,[15][16][17]. Therefore, genetic engineering revealed differences between natural plant accumulators and non-accumulators of GB with regard to their respective capacities to regulate the Cho and the connected PC biosynthesis fluxes.…”