The Arabidopsis trehalose-6-P synthase AtTPS1 gene is a regulator of glucose, abscisic acid, and stress signaling

Abstract

In Arabidopsis (Arabidopsis thaliana), trehalose is present at almost undetectable levels, excluding its role as an osmoprotectant. Here, we report that overexpression of AtTPS1 in Arabidopsis using the 35S promoter led to a small increase in trehalose and trehalose-6-P levels. In spite of this, transgenic plants displayed a dehydration tolerance phenotype without any visible morphological alterations, except for delayed flowering. Moreover, seedlings overexpressing AtTPS1 exhibited glucose (Glc)- and abscisic acid (ABA)-insensitive phenotypes. Transgenic seedlings germinated on Glc were visibly larger with green well-expanded cotyledonary leaves and fully developed roots, in contrast with wild-type seedlings showing growth retardation and absence of photosynthetic tissue. An ABA dose-response experiment revealed a higher germination rate for transgenic plants overexpressing AtTPS1 showing insensitive germination kinetics at 2.5 mum ABA. Interestingly, germination in the presence of Glc did not trigger an increase in ABA content in plants overexpressing AtTPS1. Expression analysis by quantitative reverse transcription-PCR in transgenic plants showed up-regulation of the ABI4 and CAB1 genes. In the presence of Glc, CAB1 expression remained high, whereas ABI4, HXK1, and ApL3 levels were down-regulated in the AtTPS1-overexpressing lines. Analysis of AtTPS1 expression in HXK1-antisense or HXK1-sense transgenic lines suggests the possible involvement of AtTPS1 in the hexokinase-dependent Glc-signaling pathway. These data strongly suggest that AtTPS1 has a pivotal role in the regulation of Glc and ABA signaling during vegetative development.

Figure 1.

Semiquantitative RT-PCR analysis of AtTPS1 expression. AtTPS1 transcript accumulation in wild-type and 10 independent transgenic lines. APT1 was used as a constitutive control. Western-blot analysis of AtTPS1 protein accumulation in wild-type and transgenic lines. SDS-PAGE showing Rubisco protein as a constitutive control. Graphics represent the quantification of the shown bands normalized according to the corresponding controls (APT1 and Rubisco).

Figure 2.

Stress-tolerance analysis of AtTPS1-overexpressing plants. Four-week-old 12.3 transgenic line (A and B) and wild-type plants (C and D) were water deprived until desiccation (A and C) and then rehydrated for 24 h (B and D). Kinetics of RWC of 10 individuals of 12.3 (○), 5.4 (□), and wild-type (⋄) plants (E). The SGWC of 5 individuals of transgenic lines 12.3 (white bars), 5.4 (black bars), and wild type (gray bars) was determined at the beginning and end of the experiment (E, inset).

Figure 3.

Glc sensitivity of 35S∷AtTPS1 plants. Phenotype of 7-d-old wild-type (A) and 12.3 transgenic-line (B) seedlings growing on MS supplemented with 6% Glc. Germination kinetics of transgenic lines overexpressing AtTPS1 in comparison to wild type and abi4 mutant growing on MS media alone (C) or on MS supplemented with 6% Glc (D). Germination was defined as complete protrusion of the radicule. The data are the mean of 3 independent experiments evaluating 100 seeds per data point. Error bar represents sd. □, Line 12.3; ▵, line 5.4; ⋄, wild-type seedlings; and ○, abi4 mutant.

Figure 4.

ABA sensitivity of 35S∷AtTPS1 plants. A, Germination dose response after 10 d on MS media supplemented with 2.5, 5, 10, or 20 μm ABA. Representative lines 12.3 (□) and 5.4 (▵) are shown in comparison to wild-type seedlings (⋄), and abi4 mutant (○). B, Germination kinetics of plants growing on MS media containing 2.5 μm ABA. Lines 12.3 (black bars) and 5.4 (gray bars) are shown in comparison to wild-type seedlings (white bars) and abi4 mutant (dashed bars). Germination was defined as complete protrusion of the radicule. The data are the mean of 3 independent experiments evaluating 100 seeds per data point. Error bar represents sd. Phenotype of 7-d-old wild-type (C) and 12.3 transgenic-line seedlings (D) growing on MS supplemented with 2.5 μm ABA.

Figure 5.

Quantitative PCR analysis of selected genes expressed in 35S∷AtTPS1 plants. Relative expression levels measured by QPCR in response to 6 h of treatment with 7% Glc or mannitol (Control) of transgenic lines 12.3, 5.4, and 4.4 and wild-type seedlings. The data were obtained from three biologically independent experiments. Detector probes: AtTPS1, ABI4, HXK1, ApL3, and CAB1.

Figure 6.

AtTPS1 expression analysis in HXK1-sense or -antisense plants. Semiquantitative RT-PCR analysis in 7-d-old wild-type (Wt), antisense (AS) or sense (S) HXK1 transgenic lines grown on MS alone or supplemented with 6% Glc. Graphics represent the quantification of the shown bands normalized according to APT1 expression.