Expression of the Arabidopsis mutant ABI1 gene alters abscisic acid sensitivity, stomatal development, and growth morphology in gray poplars

Abstract

The consequences of altered abscisic acid (ABA) sensitivity in gray poplar (Populus x canescens [Ait.] Sm.) development were examined by ectopic expression of the Arabidopsis (Arabidopsis thaliana) mutant abi1 (for abscisic acid insensitive1) gene. The expression resulted in an ABA-insensitive phenotype revealed by a strong tendency of abi1 poplars to wilt, impaired responsiveness of their stomata to ABA, and an ABA-resistant bud outgrowth. These plants therefore required cultivation under very humid conditions to prevent drought stress symptoms. Morphological alterations became evident when comparing abi1 poplars with poplars expressing Arabidopsis nonmutant ABI1 or wild-type plants. abi1 poplars showed increased stomatal size, enhanced shoot growth, and retarded leaf and root development. The increased stomatal size and its reversion to the size of wild-type plants by exogenous ABA indicate a role for ABA in regulating stomatal development. Enhanced shoot growth and retarded leaf and root development support the hypothesis that ABA acts independently from drought stress as a negative regulator of growth in shoots and as a positive regulator of growth in leaves and roots. In shoots, we observed an interaction of ABA with ethylene: abi1 poplars exhibited elevated ethylene production, and the ethylene perception inhibitor Ag(+) antagonized the enhanced shoot growth. Thus, we provide evidence that ABA acts as negative regulator of shoot growth in nonstressed poplars by restricting ethylene production. Furthermore, we show that ABA has a role in regulating shoot branching by inhibiting lateral bud outgrowth.

Figure 1.

Stable transformation of Arabidopsis genes abi1 and ABI1 in poplar and their ectopic expression. A, Amplification of abi1/ABI1 transgenes (analysis of five independent abi1/ABI1 transformants) via PCR with ABI1-specific primers. wt, Wild-type control. B, Immunoblot analysis of abi1-GUS and ABI1-GUS protein expression (analysis of three independent abi1/ABI1 transformants) with anti-GUS antibody. C, Histochemical detection of abi1-GUS and ABI1-GUS protein expression (analysis of three independent abi1/ABI1 transformants). [See online article for color version of this figure.]

Figure 2.

Effect of ABI1 and abi1 expression on ABA promotion of stomatal closure. Black box, control; gray box, 200 μm ABA. Values are mean ± se (n = 10 leaves/60 stomata per leaf, P ≤ 0.05*/0.005**, Student's t test). wt, Wild-type control.

Figure 3.

Lateral bud outgrowth of short shoot segments grown for 3 weeks on half-concentrated MS medium supplemented with ABA (50 μm) or without ABA. A and B, Poplars expressing abi1. Outgrowth of lateral buds is not inhibited by ABA. C and D, Poplars expressing ABI1. Outgrowth of lateral buds is inhibited by ABA. E and F, Wild-type (wt) poplars. Outgrowth of lateral buds is inhibited by ABA (arrows point to old petioles still clinging on the stem axis; asterisks indicate old petioles separated from the stem axis). Inserts in D and F show dormant lateral buds. [See online article for color version of this figure.]

Figure 4.

Typical stomata in wild-type (wt), ABI1, and abi1 poplars (leaf tissue double stained with anilin blue and safranin; bar 10 μm). [See online article for color version of this figure.]

Figure 5.

Aperture of fully opened stomata of abi1 poplars grown for 5 weeks on half-concentrated MS medium supplemented initially with 50 μm ABA (gray box) or without ABA (black box). Values are mean ± se (n = 10 leaves, P ≤ 0.005**, Student's t test).

Figure 6.

Growth habitus of poplar plants grown for 45 d on half-concentrated MS medium with high air humidity (RH >90%). A, Poplars expressing abi1 with elongated shoots, small leaves, and less-developed roots. B, Poplars expressing ABI1 with short shoots, large leaves, and well-developed roots. C, Wild-type poplars with stunted shoots, large leaves, and well-developed roots. [See online article for color version of this figure.]