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. 2006 Jan;140(1):115-26.
doi: 10.1104/pp.105.070128. Epub 2005 Dec 9.

ABA-hypersensitive germination3 encodes a protein phosphatase 2C (AtPP2CA) that strongly regulates abscisic acid signaling during germination among Arabidopsis protein phosphatase 2Cs

Tomo Yoshida  1 , Noriyuki Nishimura, Nobutaka Kitahata, Takashi Kuromori, Takuya Ito, Tadao Asami, Kazuo Shinozaki, Takashi Hirayama
Affiliations

ABA-hypersensitive germination3 encodes a protein phosphatase 2C (AtPP2CA) that strongly regulates abscisic acid signaling during germination among Arabidopsis protein phosphatase 2Cs

Tomo Yoshida et al. Plant Physiol. 2006 Jan.

Abstract

The phytohormone abscisic acid (ABA) regulates physiologically important developmental processes and stress responses. Previously, we reported on Arabidopsis (Arabidopsis thaliana) L. Heynh. ahg mutants, which are hypersensitive to ABA during germination and early growth. Among them, ABA-hypersensitive germination3 (ahg3) showed the strongest ABA hypersensitivity. In this study, we found that the AHG3 gene is identical to AtPP2CA, which encodes a protein phosphatase 2C (PP2C). Although AtPP2CA has been reported to be involved in the ABA response on the basis of results obtained by reverse-genetics approaches, its physiological relevance in the ABA response has not been clarified yet. We demonstrate in vitro and in vivo that the ahg3-1 missense mutation causes the loss of PP2C activity, providing concrete confirmation that this PP2C functions as a negative regulator in ABA signaling. Furthermore, we compared the effects of disruption mutations of eight structurally related PP2C genes of Arabidopsis, including ABI1, ABI2, HAB1, and HAB2, and found that the disruptant mutant of AHG3/AtPP2CA had the strongest ABA hypersensitivity during germination, but it did not display any significant phenotypes in adult plants. Northern-blot analysis clearly showed that AHG3/AtPP2CA is the most active among those PP2C genes in seeds. These results suggest that AHG3/AtPP2CA plays a major role among PP2Cs in the ABA response in seeds and that the functions of those PP2Cs overlap, but their unique tissue- or development-specific expression confers distinct and indispensable physiological functions in the ABA response.

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Figures

Figure 1.
Figure 1.
ABA-hypersensitive phenotype of ahg3-1. A and C, Germination efficiencies (radicle emergence); B and D, postgermination growth efficiencies (green cotyledons) of wild-type (black squares) and ahg3-1 (black triangles) seeds in the presence of 0.3 μm ABA for 7 d after stratification (A and B) or seeds in the presence of various concentrations of ABA at 3 d (C) or 7 d (D) after stratification. E and F, Dormancy of wild-type (black symbols) and ahg3-1 (white symbols) seeds; triangles, squares, and circles indicate seeds imbibed for 0, 2, and 4 d at 4°C, respectively. In A to F, averages of three independent experiments are shown with sds. Approximately 50 seeds were used in each experiment. G, Endogenous ABA content of dry and imbibed seeds (4°C for 4 d) of wild type (white bars) or ahg3-1 (black bars). Averages of three independent experiments are shown with sds. H, Wild-type and ahg3-1 seedlings grown on MS plates with or without 0.3 μm ABA for 7 d after stratification.
Figure 2.
Figure 2.
Identification of AHG3 by map-based gene cloning. A, Schematic representations of mapping and structure of AHG3. The exon-intron organization of AHG3 is shown. Mutation sites of ahg3-1 and ahg3-2 (T-DNA insertion) are indicated. B, Comparison of the amino acid changes caused by ahg3-1 and abi1-1R6. White letters indicate conserved amino acid residues. C, RNA gel-blot analysis of AHG3 in wild type, ahg3-1, and ahg3-2. D, ABA hypersensitivity of ahg3-2. Seeds were sown on a MS plate containing 0 or 0.3 μm ABA and grown for 7 d. The percentages of seedlings with green cotyledons are shown. Averages of three independent experiments are shown with sds. Approximately 50 seeds were used in each experiment. E, Amino acid sequence of the ahg3G145D artificial mutation similar to abi1-1. White letters indicate conserved amino acid residues. F, Coomassie Blue staining of GST-fusion proteins used in the in vitro phosphatase assay. Each lane contains approximately 1 μg of protein. G, In vitro phosphatase activity of recombinant proteins. GST-AHG3 (20 ng; black squares), GST-ahg3-1 (100 ng; black triangles), and GST-ahg3G145D (100 ng; white circles) were incubated with 32P-labeled casein in the presence of 2 μm okadaic acid. PP2C activity is expressed in picomoles of released 32Pi. Values shown are the means of duplicate assays. The error bar is not shown if it is smaller than the symbol size. H, Complementation analysis of AHG3. Seeds of transgenic ahg3-1 plants possessing the genomic AHG3 clone or vector-control plants were germinated and grown in the presence of 0.3 μm ABA for 7 d. Averages of three independent experiments are shown with sds. Approximately 50 seeds were used in each experiment.
Figure 3.
Figure 3.
T-DNA or Ds insertional mutations of PP2C genes. Schematic representation of the T-DNA or Ds insertion site of PP2C mutants used in this study. Gray box and striped box indicate T-DNA and Ds transoposon, respectively. White box, black box, and horizontal thick line indicate 5′- or 3′-untranslated region, exon, and intron, respectively. Number indicates the nucleotide position from the cDNA start point. There is no cDNA reported for At2g29380.
Figure 4.
Figure 4.
ABA sensitivity of ahg3-2 was stronger among ABA-related PP2C insertion mutants. A, Seedlings of wild-type plants and PP2C insertion mutants germinated in the presence of 0 or 0.3 μm ABA. Photographs were taken after a 7-d incubation. B, Postgermination growth efficiency of wild-type plants and PP2C insertion mutants. About 50 seeds were sown on MS plates containing various concentrations of ABA and incubated for 7 d. C, Chlorophyll content of T-DNA insertion lines (top) and Ds transposon insertion lines (bottom). Seeds were sown on MS plates containing various concentrations of ABA and incubated for 10 d. In B and C, averages of three independent experiments are shown with sds. Approximately 50 seeds were used in each experiment.
Figure 5.
Figure 5.
Expression of PP2C genes in seeds. A, Northern-blot analysis of PP2C genes implicated in the ABA response in seeds and adult plants. Each lane contains approximately 10 μg of total RNA extracted from dry seeds (a), from seeds grown on MS plates for 1 d (b) and 3 d (c), from seeds grown on MS plates containing 1.0 μm ABA for 3 d (d), or from 3-week-old plants treated with 100 μm ABA for 0 (e), 1 (f), 2 (g), 5 (h), 10 (i), and 24 h (j), respectively, after preincubation in water for 2 h. The β-TUBULIN gene was used as a loading control. B, Relationship between overall structure, mRNA level in seeds, and effect of mutation on ABA sensitivity of PP2Cs. A phylogenetic tree was constructed using ClustalW. The ABA sensitivity of insertion mutants and mRNA levels in imbibed seeds are summarized. a and b, This study; c, according to the AtGenExpress microarray database.
Figure 6.
Figure 6.
In planta effects of overexpression and abi1-1-type mutation of AHG3. Effect of overexpression of AHG3/AtPP2CA (A) or ahg3G145D (B) on ABA sensitivity during germination. Seeds were sown and grown on MS plates containing 0.3 μm ABA for 7 d.
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