Overexpression of the trehalase gene AtTRE1 leads to increased drought stress tolerance in Arabidopsis and is involved in abscisic acid-induced stomatal closure

Abstract

Introduction of microbial trehalose biosynthesis enzymes has been reported to enhance abiotic stress resistance in plants but also resulted in undesirable traits. Here, we present an approach for engineering drought stress tolerance by modifying the endogenous trehalase activity in Arabidopsis (Arabidopsis thaliana). AtTRE1 encodes the Arabidopsis trehalase, the only enzyme known in this species to specifically hydrolyze trehalose into glucose. AtTRE1-overexpressing and Attre1 mutant lines were constructed and tested for their performance in drought stress assays. AtTRE1-overexpressing plants had decreased trehalose levels and recovered better after drought stress, whereas Attre1 mutants had elevated trehalose contents and exhibited a drought-susceptible phenotype. Leaf detachment assays showed that Attre1 mutants lose water faster than wild-type plants, whereas AtTRE1-overexpressing plants have a better water-retaining capacity. In vitro studies revealed that abscisic acid-mediated closure of stomata is impaired in Attre1 lines, whereas the AtTRE1 overexpressors are more sensitive toward abscisic acid-dependent stomatal closure. This observation is further supported by the altered leaf temperatures seen in trehalase-modified plantlets during in vivo drought stress studies. Our results show that overexpression of plant trehalase improves drought stress tolerance in Arabidopsis and that trehalase plays a role in the regulation of stomatal closure in the plant drought stress response.

Figure 1.

Characterization of AtTRE1-modified lines. A, Immunoblot (IB) of AtTRE1 accumulation (approximately 64 kD) in protein extracts from seedlings, rosettes, flowers, and siliques and the corresponding Coomassie Brilliant Blue (CBB) stain showing the Rubisco large subunit as a constitutive control. B, Growth of trehalase mutants and wild-type (Wt) seedlings on 0.5× MS medium with and without 25 mm trehalose.

Figure 2.

A, Trehalase-modified plants after 20 d of water restriction, followed by a recovery period of 48 h upon rewatering. Five individual plants of each transgenic line were ordered from left to right according to their decreasing ability to survive upon drought stress. Wt, Wild type. B, Kinetics of water loss from detached leaves. Left panel, wild-type Ler (black circles) and Attre1-1 (white circles); right panel, wild-type Col-0 (black squares), Attre1-2 (white squares), Attre1-3^OE (black triangles), 9.1 (white triangles), and 35S::treF (black diamonds). Water loss is expressed as the percentage of initial fresh weight (FW). Regression analysis (Student’s t test) showed significant differences between the wild-type Ler curve and the Attre1-1 curve (P < 0.0001) and between the wild-type Col-0 curve and the Attre1-2, Attre1-3^OE, 9.1, and 35S::treF curves (P < 0.075). Values represent averages ± se (n = 3 batches containing three leaves each).

Figure 3.

Histochemical localization of GUS activity in pTRE1::GUS-GFP lines during germination at the stages of root protrusion and hypocotyl stretching (A), in 5-d-old seedlings (B), in 10-d-old seedlings (C and D), in 5-week-old bolted plants (E), in the mature, second real leaf from a 5-week-old plant (F), in flower buds (G), in the flower stem, in cauline leaves, and in flower buds on secondary inflorescences (H), in the inflorescence (I), and in a developing seed at early maturation stage (J).

Figure 4.

Leaf stomatal features. A, Stomatal pore sizes without ABA (black bars) and with 20 µm ABA (white bars). Stomatal apertures of the trehalase-modified lines within each treatment are significantly different from the wild type (Wt Ler and Wt Col-0) at P < 0.01 (*) and P < 0.0001 (**) by Student’s t test. Values represent averages ± se (n = 80). B, Relative AtTRE1 expression in wild-type seedlings treated for 60 min with 10 µm ABA is significantly increased compared with the solvent control (0 µm ABA) at P < 0.05 (*) by Student’s t test. Values represent averages ± sd (n = 3; each replicate represents a pool of at least 20 seedlings). C, Stomatal index of the trehalase-modified lines is significantly different from the wild-type index (Wt Ler and Wt Col-0) at P < 0.05 (*), P < 0.001 (**), and P < 0.0001 (***) by Student’s t test on data following the arcsine transformation. Values are means ± se (n = 60).

Figure 5.

Transpiration of trehalase-modified seedlings restricted in water for 4 d. A, False-color infrared images of the drought-stressed plantlets. B, Normalized leaf temperatures of the transgenic seedlings as calculated from the quantification of three infrared images (approximately 3,000 square pixels) as shown in A are significantly different from wild-type (Wt) Ler and Col-0 at P < 0.0001 (*; Z score). Values are weighted means ± sd.