An Arabidopsis inositol 5-phosphatase gain-of-function alters abscisic acid signaling

Abstract

Signals can be perceived and amplified at the cell membrane by receptors coupled to the production of a variety of second messengers, including inositol 1,4,5-trisphosphate (IP3). We previously have identified 15 putative inositol 5-phosphatases (5PTases) from Arabidopsis and shown that At5PTase1 can hydrolyze IP3. To determine whether At5PTase1 can terminate IP3-mediated signaling, we analyzed transgenic plants ectopically expressing At5PTase1. Stomata from leaves of At5PTase1 transgenic plants were abscisic acid (ABA) and light insensitive, and ABA induction of genes was delayed. Quantification of IP3 in plants exposed to ABA indicated that ABA induced two IP3 increases in wild-type plants. Both of these IP3 increases were reduced in At5PTase1 transgenic plants, indicating that IP3 may be necessary for stomatal closure and temporal control of ABA-induced gene expression. To determine if ABA could induce expression of At5PTase1, we examined RNA and protein levels of At5PTase1 in wild-type plants exposed to ABA. Our results indicate that At5PTase1 is up-regulated in response to ABA. This is consistent with At5PTase1 acting as a signal terminator of ABA signaling.

Figure 1.

Protein-blot analysis of transgenic lines expressing the At5PTase1 transgene. Equal amounts of crude protein extract from leaves of wild type (WT) or three independent lines of At5PTase1transgenic plants (1–3) were separated by SDS-PAGE, blotted onto nitrocellulose, and probed with a rabbit antibody specific for At5PTase1 protein as described in “Materials and Methods.” Arrows indicate molecular mass standards.

Figure 2.

At5PTase1 transgenic stomata are altered in their responses to ABA and light but not fusicoccin. Leaves from WT and three independent lines of At5PTase1 transgenic plants (1–3) were removed from the plant and incubated in the indicated solutions (see “Materials and Methods”). Epidermal fragments generated by blender treatment were imaged to determine the average stomatal apertures as described in “Materials and Methods.” A, Leaves were incubated in a perfusion solution for 2 h (white bars) and then exposed to 100 μm ABA for 2 h (black bars). B, Leaves were incubated in a perfusion solution for 2 h (white bars) and then exposed to 15 μm fusicoccin for 2 h (black bars). C, Leaves were incubated in 10 mm MES, 50 mm KCl, and 100 μm ABA (pH 7.0) for 2 h in the dark (black bars) and then exposed to light for 2 h (gray bars). Separate leaves were incubated in perfusion solution under constant light for 2 h (white bars). Error bars = se from three independent experiments.

Figure 3.

Ectopic expression of At5PTase1 suppresses ABA-stimulated IP₃ accumulation. Five-day-old WT (white diamonds) and At5PTase1 transgenic seedlings (black squares) were treated with 100 μm ABA and harvested at the indicated times. IP₃ was quantified from frozen ground tissue as described in “Materials and Methods.” A, Five-minute time course. B, One hundred-twenty-minute time course. Error bars = mean se from three independent experiments.

Figure 4.

Expression of ABA inducible genes is altered in At5PTase1 transgenic plants. WT (5 d) or At5PTase1 transgenic (Trans) seedlings were treated with ABA and harvested at the indicated times. Total RNA was isolated, and RT-PCR reactions were performed with Kin 1-, COR15a-, Kin 2-, or actin-specific primers as described in “Materials and Methods.” Control (C) indicates seedlings treated with 0.1% (v/v) ethanol for 15 s.

Figure 5.

Germination of At5PTase1 transgenic seed in the presence of ABA. WT seed (black squares) and At5PTase1 transgenic seed (white circles) were germinated with no ABA present. WT seed (black triangles) and At5PTase1 transgenic seed (black circles) were germinated in the presence of 2.5 μm ABA. No differences in germination were observed at 0.1 or 0.25 μm ABA (data not shown), and no germination was observed at concentrations of ABA above 5 μm (data not shown). Error bars = mean ± sd values from three independent experiments during a 6-d period.

Figure 6.

ABA treatment alters At5PTase1 expression in seedlings. Five- to 7-d-old WT seedlings were treated with 100 μm ABA and harvested at the indicated times. A, Total RNA was harvested and used in RT-PCR experiments with At5PTase1- and actin-specific primers as described in the “Materials and Methods.” -, No template; +, control cDNA template. B and C, Protein extracts were generated, followed by SDS-PAGE and protein gel-blot analysis as described in “Materials and Methods.” Arrows indicate molecular mass standards.