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CELL BIOLOGY AND SIGNAL TRANSDUCTION

ABA-Hypersensitive Germination3 Encodes a Protein Phosphatase 2C (AtPP2CA) That Strongly Regulates Abscisic Acid Signaling during Germination among Arabidopsis Protein Phosphatase 2Cs^1,[W]

International Graduate School of Arts and Sciences, Yokohama City University, Tsurumi, Yokohama 230–0045, Japan (T.Y., N.N., T.H.); Plant Molecular Biology, The Institute of Physical and Chemical Research (RIKEN) Tsukuba Institute, Tsukuba, Ibaraki 305–0074, Japan (N.N., T.I., K.S., T.H.); Laboratory of Cellular Biochemistry, RIKEN Wako Institute, Wako, Saitama 351–0198, Japan (N.K., T.A.); and Plant Functional Genomics Research Group, Genomic Sciences Center, RIKEN Yokohama Institute, Tsurumi, Yokohama 230–0045, Japan (T.K., K.S., T.H.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
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.

Considerable efforts to understand the ABA response mechanisms, using forward- and reverse-genetics approaches mainly with Arabidopsis (Arabidopsis thaliana) L. Heynh., have identified a number of components implicated in the ABA-signaling pathway (for review, see Finkelstein et al., 2002). ABI3, ABI4, and ABI5 encode different types of transcription factors that regulate the expression of ABA-responsive genes in seeds (Giraudat et al., 1992; Finkelstein et al., 1998; Finkelstein and Lynch, 2000). ABI5 seems to interact directly with ABI3 (Nakamura et al., 2001) and to be negatively regulated by a ubiquitin-dependent protein degradation system involving AFP, an ABI5-interacting protein (Lopez-Molina et al., 2003). ERA1, a -subunit of farnesyl transferase, is thought to act as a negative regulator of ABA signaling (Cutler et al., 1996) and to modulate the expression of ABI3 (Brady et al., 2003). HYL1, ABH1, and SAD1, isolated in studies of Arabidopsis ABA-hypersensitive mutants, encode a double-stranded RNA-binding protein, an mRNA cap-binding protein, and an Sm-like snRNP, respectively. These proteins are thought to be involved in mRNA processing of some genes implicated in the ABA response (Lu and Fedoroff, 2000; Hugouvieux et al., 2001; Xiong et al., 2001). Recently, another ABA-hypersensitive locus AHG2 was shown to encode a polyA-specific ribonuclease, indicating the involvement of mRNA degradation in ABA and stress responses (Nishimura et al., 2005). Analysis of another ABA-hypersensitive mutant, rpn10, suggests the involvement of the 26S proteasome system in ABA signaling (Smalle et al., 2003). A G-protein-coupled receptor, GCR1, and a G-protein -subunit, GPA1, are thought to be involved in ABA-mediated stomatal closure (Wang et al., 2001; Pandey and Assmann, 2004).

Protein phosphorylation and dephosphorylation are involved in ABA signaling. AAPK, an Snf-1-related protein kinase 3 (SnRK3) of Vicia faba and OST1/SnRK2E of Arabidopsis are required in the ABA response of guard cells (Li et al., 2000; Mustilli et al., 2002; Yoshida et al., 2002). PKABA1, a member of the Triticum SnRK2 family, is thought to function in ABA signaling in seeds (Johnson et al., 2002). Analysis of the rcn1 mutant suggests that a protein phosphatase 2A functions as a positive signal transducer of early ABA signaling (Kwak et al., 2002). In addition, several protein phosphatase 2Cs (PP2Cs) are deeply involved in ABA signaling (Rodriguez, 1998; Schweighofer et al., 2004). ABI1 and ABI2 encode homologous PP2Cs that had been identified in studies of two dominant ABA-insensitive mutants, abi1-1 and abi2-1 (Leung et al., 1994, 1997; Meyer et al., 1994). Intragenic suppressors of abi1-1 and abi2-1 were isolated and shown to be ABA hypersensitive, indicating their functions as negative regulators of the ABA response (Gosti et al., 1999; Merlot et al., 2001). Results consistent with this idea were obtained in studies of Arabidopsis HAB1/AtP2C-HA and Fagus sylvatica FsPP2C1 (Gonzalez-Garcia et al., 2003; Leonhardt et al., 2004; Saez et al., 2004). AtPP2CA, another Arabidopsis PP2C, was shown to block ABA signal transduction when transiently expressed in maize (Zea mays) mesophyll protoplasts (Sheen, 1998). Downregulation of AtPP2CA by an antisense gene accelerated plant development and led to freezing tolerance (Tahtiharju and Palva, 2001). These results indicate the role of AtPP2CA as a negative regulator of the ABA response. However, the physiological relevance of AtPP2CA in the ABA response has not been clarified because of the lack of a loss-of-function mutant to date.

Here, we describe the study of a novel Arabidopsis ABA-hypersensitive mutant, ABA-hypersensitive germination3 (ahg3), which has been isolated by screening with an ABA analog (Nishimura et al., 2004). AHG3 is identical to AtPP2CA and ahg3-1 is a type of loss-of-function mutation. We examined the ABA sensitivity in germinating seeds of T-DNA or Dissociation (Ds) transposon insertion mutants of ABA-implicated PP2Cs and found that the mutation of AHG3/AtPP2CA had the strongest effect. Our results confirm the presumption that AHG3/AtPP2CA functions as a negative regulator of the ABA-signaling pathway and suggest that AHG3/AtPP2CA has a major role in ABA signaling in seed germination and early growth.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Physiological Characterization of ahg3-1

ahg3-1 is an ABA-hypersensitive mutant that germinates and grows poorly in the presence of ABA (Nishimura et al., 2004). To elucidate the function of AHG3 in the ABA response, we investigated the efficiency of radicle emergence and early growth of this mutant in the presence of various concentrations of ABA. ahg3-1 plants showed a slight, but clear, reduction in radicle emergence in the presence of exogenous ABA (Fig. 1, A and C ). By contrast, they showed strong growth retardation after germination (Fig. 1, B, D, and H). We examined the effect of seed stratification on the germination of ahg3-1. As shown in Figure 1, E and F, without stratification the ahg3-1 seeds germinated and grew poorly. In contrast, stratification treatments dramatically improved those abilities.

View larger version (27K): [in this window] [in a new window]   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.

The ahg3-1 plants grew slightly slower than wild-type plants on soil, but the final plant size was the same as the wild type. The bolting time was also slightly delayed; the wild-type plants started to bolt at 22 to 25 d after sowing, but ahg3-1 plants needed 2 to 3 d longer. However, the numbers of rosette leaves when bolting started were almost the same in wild-type and ahg3-1 plants (wild type, 12.5 ± 0.85; ahg3-1, 12.7 ± 0.82; n = 10), suggesting that the delay in bolting of ahg3-1 is due not to abnormal flowering control but to slower growth. These results suggest that growth retardation is due not to altered ABA sensitivity in later growth stages but presumably to slower germination of agh3-1 (Fig. 1, E and F). We examined the ABA sensitivity of rosette plants. Seven-day-old seedlings were placed on an ABA-containing plate and grown for 10 d. The ahg3-1 plants grew identically to the wild type (data not shown), implying that ahg3-1 does not have a detectable ABA-hypersensitive phenotype in the adult stage under our experimental conditions. In the previous study, we examined the expression of ABA- and stress-inducible genes—RD29A, RD29B, P5CS, and RAB18—by RNA gel-blot analysis and found no significant changes in ahg3-1 (Nishimura et al., 2004). In this study, we examined those in more detail and obtained the same results (Supplemental Fig. 1). These results suggest that ahg3-1 does not affect ABA sensitivity so strongly in adult plants under the imposed conditions.

Double-Mutant Analysis

To characterize ahg3-1 genetically, we performed epistatic analysis. ahg3-1 plants were crossed with abi1-1, abi2-1, abi3-1, abi4-1, and abi5-1 plants, and double-mutant lines were obtained from F₂ progeny. We examined the early growth efficiencies of those lines on Murashige and Skoog (MS) plates containing 0.3 or 3.0 µM ABA (Table I). The ahg3-1abi1-1, ahg3-1abi4-1, and ahg3-1abi5-1 double mutants showed less ABA resistance than the parental monogenic mutant, suggesting additive effects between ahg3-1 and these abi mutations. By contrast, ahg3-1 had little or no effect on the ABA sensitivity of abi2-1 and abi3-1. The germination efficiency of abi2-1 was reduced by 3.0 µM ABA. The ahg3-1 mutation did not seem to enhance this ABA effect. Even 3.0 µM ABA had only a subtle effect on the germination efficiencies of both abi3-1 and ahg3-1abi3-1. We obtained almost the same results using independent double-mutant lines, suggesting that the effect of the ecotype background was negligible (data not shown). These results might indicate that AHG3 functions at or upstream of ABI2 and ABI3 if the ABA-signaling pathway is linear.

We identified the AHG3 gene by map-based cloning. The AHG3 locus was mapped near the marker nga162 on chromosome 3 (Nishimura et al., 2004). We analyzed 1,746 chromosomes of F₂ plants obtained from an ahg3-1 x Landsberg erecta (Ler) test cross and narrowed the AHG3 locus to a region spanning about 70 kb (Fig. 2A ). By determining the nucleotide sequence of this region, we found a base conversion from G to A in the third exon of At3g11410 (Arabidopsis Genome Initiative, 2000), which encodes a PP2C, AtPP2CA (Kuromori and Yamamoto, 1994). This mutation causes the amino acid change of Gly-287 to Glu in the predicted gene product. Interestingly, the ahg3-1 mutation occurred at the corresponding amino acid residue of the abi1-1R6 mutation site, one of the intragenic suppressor mutants of abi1-1 (Fig. 2B; Gosti et al., 1999). We obtained a T-DNA insertion allele of AHG3 from the SALK T-DNA lines (SALK_028132; Alonso et al., 2003). The T-DNA was inserted in the end of the second exon; we named this line ahg3-2 (Fig. 2A). Northern-blot analysis of ahg3-2 failed to detect the correct size of AHG3 mRNAs, suggesting that ahg3-2 is a null allele (Fig. 2C). ahg3-2 showed ABA-hypersensitive phenotypes in germination and early growth as strong as those of ahg3-1 (Fig. 2D).

View larger version (39K): [in this window] [in a new window]   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.

To confirm that At3g11410 is AHG3, we generated a transgenic ahg3-1 plant possessing a wild-type At3g11410 gene. Transgenic plants showed normal early growth in the presence of ABA (Fig. 2H). From these results, we concluded that At3g11410 is AHG3. Taken together, these results strongly suggest that the defect in AHG3/AtPP2CA caused the ABA-hypersensitive phenotype and that AHG3/AtPP2CA is indeed a negative regulator of ABA signaling.

ahg3-2 Showed the Strongest ABA Hypersensitivity among T-DNA Insertion Mutants of ABA-Related PP2Cs

At least four Arabidopsis PP2C genes, ABI1, ABI2, HAB1/AtP2C-HA, and AHG3/AtPP2CA, have been shown to be ABA inducible (Rodriguez, 1998) and are thought to function in the ABA-signaling pathway. So far, there is very little information about the redundant or distinct roles of their proteins. It is of interest to clarify whether our isolation of ahg3-1 as an ABA-hypersensitive mutant reflects a specific function of AHG3/AtPP2CA or happened just by chance. We obtained T-DNA or Ds transposon insertion mutants of PP2C genes closely related to AHG3/AtPP2CA (Alonso et al., 2003; Kuromori et al., 2004; Ito et al., 2005): ABI1 (abi1td, SALK_076309; abi1ds, 13-4144-1); ABI2 (abi2td, SALK_015166); HAB1/AtP2C-HA (hab1-1, SALK_002104; Leonhardt et al., 2004; Saez et al., 2004); HAB2 (hab2ds, 15-1860-1); At2g29380 (at2g29380td, SALK_033011); At1g07430 (at1g07430ds, 53-2196-1); and At5g59220 (at5g59220ds, 53-2870-1; Fig. 3 ). The T-DNA or Ds insertion sites of abi1td, abi1ds, hab1-1, at1g07430ds, and at5g59220ds are located in the coding regions, indicating that these are null mutations. The insertion sites of hab2ds and at2g29380td are located in an intron and the 5'-untranslated region, respectively. The expression of these genes was undetectable by reverse transcription (RT)-PCR, indicating that these also are null mutations (see Supplemental Fig. 2). The T-DNA insertion site of abi2td is the C terminus of the ABI2-coding region. We could not conclude whether this mutation was null or not. However, the phenotype (see below) is stronger than that of the abi2-1 revertant mutant reported previously (Merlot et al., 2001). Therefore, it is plausible that the abi2td mutation also reduced the function of ABI2 to a very low level. The homozygosity of the T-DNA or Ds insertion was confirmed by examining the genome structure by a PCR-based method.

View larger version (19K): [in this window] [in a new window]   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.

View larger version (42K): [in this window] [in a new window]   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.

The results described above raised the question of what determines the major role in seeds of AHG3/AtPP2CA among related PP2Cs. The total amino acid sequence similarity cannot account for it, because the insertion mutations of At2g29380, At1g07430, and At5g59220, which are closely related to AHG3/AtPP2CA (Fig. 5B ), did not have any effect. According to published DNA microarray experiment data (Nakabayashi et al., 2005; AtGenExpress), the mRNA levels of these PP2C genes are considerably different between dry and imbibed seeds. Therefore, it might be possible that the differences in the expression of these genes explain the different contributions of these PP2Cs to ABA signaling in seeds. We performed RNA gel-blot analyses to investigate the mRNA levels of AHG3/AtPP2CA and several other PP2C genes in seeds. Gene-specific probes were designed from dissimilar N-terminal regions, and the signal strength of each probe was validated by genome dot-blot hybridization (see Supplemental Fig. 3). As expected, in dry seeds, the expression of AHG3/AtPP2CA was much stronger than those of ABI1, HAB1, and HAB2 (Fig. 5A, lane a). The mRNA level of AHG3/AtPP2CA did not seem to change during stratification and germination (Fig. 5A, lanes b and c; Supplemental Fig. 4). Using the same probes, we examined the mRNA levels in adult plants. These mRNA levels were up-regulated by ABA treatment and seemed similar among the PP2C genes. These results show that the expression level of AHG3/AtPP2CA is significantly higher in seeds, but the same as other PP2C genes in adult plants.

View larger version (48K): [in this window] [in a new window]   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 -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.

If AHG3/AtPP2CA is a negative regulator of the ABA response in seeds, overexpression of AHG3/AtPP2CA should confer ABA insensitivity. To evaluate this idea, we constructed transgenic plants possessing the P_35S:AHG3/AtPP2CA fusion gene. T₂ seeds obtained from resultant transgenic lines expressing the transgene showed considerable ABA insensitivity (Fig. 6A ), confirming that AHG3/AtPP2CA functions as a negative regulator.

View larger version (60K): [in this window] [in a new window]   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.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Isolation of the ahg3-1 Mutant

We characterized the Arabidopsis ahg3-1 line, which displays strong ABA hypersensitivity during germination. We found that the AHG3 gene is identical to AtPP2CA, which encodes a PP2C. AtPP2CA was originally cloned by transcomplementation using a fission yeast (Schizosaccharomyces pombe) mutant defective in meiosis (Kuromori and Yamamoto, 1994). Transient-assay experiments using maize mesophyll cells and studies with antisense genes implicated AtPP2CA in ABA signaling as a negative regulator (Sheen, 1998; Tahtiharju and Palva, 2001). However, its physiological relevance in the ABA response has been obscure. In this study, we clearly demonstrated that the defect in AHG3/AtPP2CA conferred ABA hypersensitivity. First, we showed that the recombinant ahg3-1 protein had undetectable PP2C activity in the in vitro assay. Second, we confirmed that ahg3-2, a null-type insertion mutant, displayed almost the same phenotype as ahg3-1, suggesting that ahg3-1 mutation results in the defect in PP2C activity in vivo. Third, we constructed transgenic plants overexpressing this gene and found that they are ABA resistant during germination. Together, these results offer conclusive evidence that AHG3/AtPP2CA functions as a negative regulator of the ABA response.

In addition, analysis of ahg3-1 offered further valuable information. So far, there are only two single missense mutations of PP2C, abi1-1 and abi2-1, which are known to cause the dominant strong ABA insensitivity. It has been explained that abi1-1 and abi2-1 are dominant-negative mutants because the mutant proteins had significantly lower PP2C activity in in vitro assay (Gosti et al., 1999), although the molecular nature of these mutations is not yet fully understood. As shown above, the ahg3-1 mutation alone causes the defect in PP2C function both in vivo and in vitro without affecting transcription (Fig. 2C). The abi1-1-type mutation in ahg3^G145D allowed AHG3 to block the ABA response in seeds despite the absence of PP2C activity in the in vitro assay, indicating the similar physiological and biochemical properties of AHG3 and ABI1. If abi1-1, abi2-1, and ahg3^G145D cause dominant-negative effects by expressing phosphatase-defective PP2C proteins, ahg3-1 would have a dominant-negative effect, but it does not. Therefore, it is more likely that a missense loss-of-function mutation of PP2C cannot confer a dominant-negative effect, and that abi1-1 and abi2-1 are, instead, gain-of-function mutations. This assumption can explain all the results except for that of the in vitro PP2C assay. That inconsistency might be due to the use of an artificial substrate. If this is the case, we should consider more carefully the results obtained using abi1-1 and abi2-1, since gain-of-function mutations do not always reflect the original gene function.

Redundant and Distinct Functions of PP2Cs

Besides AHG3/AtPP2CA, three Arabidopsis PP2Cs (ABI1, ABI2, and HAB1) have been postulated to be negative regulators of ABA signaling (Sheen, 1998; Gosti et al., 1999; Merlot et al., 2001; Leonhardt et al., 2004; Saez et al., 2004). Merlot et al. (2001) reported that ABI1 and ABI2 contribute nearly 50% of the ABA-induced PP2C activity, indicating that other PP2Cs may also be involved in redundant ABA signaling. The functional redundancy of genes often becomes an obstacle to isolating mutants. So why could we easily isolate a loss-of-function allele of AtPP2CA? The most plausible explanation is that AHG3/AtPP2CA possesses major or unique roles in the ABA response in seed germination, so the ABA-hypersensitive phenotype of ahg3-1 was not suppressed by other PP2Cs. To test this hypothesis, we performed a comparative analysis of eight PP2Cs belonging to the same group by using T-DNA or Ds insertion mutants. Most of these mutants showed ABA hypersensitivity during germination and early growth, as previously shown in hab1-1 (Saez et al., 2004), but at different intensities. In our assay, among these disruptant mutants, ahg3-2 showed the strongest ABA hypersensitivity in early growth (Fig. 4). This result indicates that AHG3/AtPP2CA is implicated more deeply in ABA signaling during germination and postgermination growth than other PP2Cs. We failed to detect any obvious phenotypes of ahg3-1 in the adult stage under our experimental conditions. Although we cannot exclude the possibility that ahg3-1 adult plants have some ABA-related phenotypes under certain conditions, it is more likely that the phenotype during germination is more significant.

So how does AHG3/AtPP2CA play a major role in seeds? AHG3/AtPP2CA was expressed more strongly in seeds than ABI1, HAB1, and HAB2, whose disruptive mutations affect ABA sensitivity during germination (Fig. 5A). We also checked the expression profiles of several PP2C genes in seeds in the AtGenExpress microarray database. Consistent with our results, according to this database, AHG3/AtPP2CA showed the strongest expression (Fig. 5B). The mechanisms that regulate PP2C are not understood at all. All plant PP2Cs examined so far are active in in vitro assays, implying that plant PP2Cs are intrinsically active. The fact that the expression of ABA-related PP2C genes is up-regulated by ABA leads to the possibility that PP2C action is regulated at the transcriptional level. We think that the higher expression of AHG3/AtPP2CA contributes to its predominant role in seeds. This idea is consistent with the lower expression of AHG3/AtPP2CA in the adult stage where we failed to detect any significant effects of ahg3-1 under our experimental conditions. ABA-related PP2Cs have shown distinct tissue-specific expression patterns (Leung et al., 1997; Rodriguez et al., 1998; Tahtiharju and Palva, 2001; Saez et al., 2004), implying that such distinct expression patterns determine the physiological functions of each ABA-related PP2C.

Substrate specificity also might determine their specific function. Thus, it is important to identify the substrate so as to understand not only the mechanism of how PP2Cs regulate ABA signaling, but also their distinct roles. Yeast (Saccharomyces cerevisiae) two-hybrid screening allowed the isolation of potassium channel AKT2 as a substrate candidate for AHG3/AtPP2CA (Vranova et al., 2001; Cherel et al., 2002). Cherel et al. (2002) described the modulation of AKT2 activity by AHG3/AtPP2CA in animal cells and the overlapped tissue-specific expression of AHG3/AtPP2CA and AKT2. However, according to the AtGenExpress microarray database, the expression of AKT2 is weaker in seeds than in other tissues, whereas that of AHG3/AtPP2CA is higher. In addition, we failed to detect any effects of the Ds insertion in an AKT2 exon (15-5406-1) on ABA sensitivity during germination (T. Yoshida, T. Kuromori, and T. Hirayama, unpublished data). Therefore, it is likely that AHG3/AtPP2CA has other substrates in seeds.

The double-mutant analysis showed that abi2-1, but not abi1-1, can suppress ahg3-1. This result indicates that abi2-1 has a stronger effect on AHG3/AtPP2CA function. This result seems consistent with the notion that ABI1 and ABI2 have different functions. However, ABI1 has no less important a role than ABI2 in ABA response in seeds because abi1-1 and abi2-1 have similar ABA insensitivity during germination, and abi1-1 revertant mutants displayed stronger ABA hypersensitivity than abi2-1 revertant mutants. Our results might reflect the stronger similarity between AHG3/AtPP2CA and ABI2 in substrate preference, spatial expression in seeds, or subcellular localization than that between AHG3/AtPP2CA and ABI1.

Physiological Function of AHG3/AtPP2CA in Seeds

As described above, AHG3/AtPP2CA functions as a negative regulator of ABA signaling in seeds. Interestingly, the endogenous ABA level was increased in ahg3-1, but returned to a normal level after a 4-d stratification (Fig. 1G). Consistent with this result, unstratified ahg3-1 seeds germinated poorly on normal media, but stratified seeds germinated as well as the wild-type seeds. These results indicate that ahg3-1 seeds tend to accumulate more ABA during seed maturation, but the down-regulation of ABA after stratification seems normal. Most genes for ABA biosynthesis are up-regulated by ABA and are regulated by positive feedback (for review, see Xiong and Zhu, 2003). Thus, the reason why ahg3-1 seeds accumulate higher levels of ABA might be the positive feedback of the ABA response. This higher accumulation of ABA in seeds contributes to ABA hypersensitivity in early growth by up-regulating or prolonging the expression of seed-specific genes (Nishimura et al., 2004). By contrast, after stratification, the endogenous ABA content of ahg3-1 dropped to normal, while its mRNA level was unchanged (Fig. 5A; Supplemental Fig. 4), indicating that AHG3 is not involved in the mechanism of ABA down-regulation in seeds.

Analysis of double mutants demonstrated that abi3-1 nearly suppressed ahg3-1, whereas abi4-1 and abi5-1 suppressed ahg3-1 more partially, indicating that ABI3 plays an important role in the ABA response downstream of AHG3/AtPP2CA. It has been proposed that the transcriptional factors ABI3, ABI4, and ABI5 regulate ABA response in seeds. They recognize different cis-elements but function together (Soderman et al., 2000). The null mutants of ABI3 cause severe defects in seed development and maturation (Nambara et al., 1992), whereas null mutations of ABI4 and ABI5 have less effect, indicating that ABI3 has the most important function among them in ABA-responsive gene expression in seed. Our results of double-mutant analysis are consistent with this idea. Recent studies showed that ABI4 is more implicated in sugar response, implying its distinct function (for review, see Gibson, 2004). ABI5 is thought to function with ABI3 (Nakamura et al., 2001). Arabidopsis has several genes for the ABI5-like protein ABF/AREB (Jakoby et al., 2002). Therefore, the reason for the partial suppression of ahg3-1 by abi5-1 might be the redundancy among these ABF/AREBs. Alternatively, it is possible that ABA signaling is not transduced by such a simple linear pathway. The additive effects of mutations may imply the higher complexity of the ABA-signaling network.

Transgenic Arabidopsis plants with lowered expression of AHG3/AtPP2CA did not display any phenotype of dehydration stress (Tahtiharju and Palva, 2001). Consistent with this result, we could not detect any significant difference in the drought response between ahg3-1 and the wild type (data not shown). Similarly, loss-of-function mutations of ABI1, ABI2, and hab1-1 did not affect transpiration rate, although abi1-1, abi2-1, and overexpression of HAB1 caused higher transpiration rates (Murata et al., 2001; Saez et al., 2004). These results indicate that these PP2Cs, including AHG3/AtPP2CA, which are up-regulated by ABA in stomata (Leonhardt et al., 2004), are implicated in ABA-mediated stomatal closure, although their functions are redundant.

In summary, by mapping of AHG3, we identified a single missense loss-of-function allele of a PP2C gene. Our data confirm the function of AHG3/AtPP2CA as a negative regulator of the ABA response. Comparative analysis of insertion mutants of eight PP2C genes suggested that AHG3/AtPP2CA is implicated deeply in the ABA response in germinating seeds. Further analyses using mutants obtained in this study will provide additional insights into the role of AHG3/AtPP2CA and other PP2Cs in ABA signaling.

Kuhn et al. (2006) have conducted an independent screen for ABA-signaling components in Arabidopsis that is similar to our research. They have characterized the same protein phosphatase AtPP2CA showing ABA insensitivity in AtPP2CA overexpressors and ABA hypersensitivity of loss-of-function mutants.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) L. Heynh. ecotype Columbia was used in this study unless otherwise indicated. Plant growth conditions have been described elsewhere (Nishimura et al., 2004).

Genetic Mapping and Identification of the AHG3 Locus

F₂ progeny obtained from a test cross between ahg3-1 and Ler were grown on MS medium containing 0.2 µM ABA for 7 d. ABA-hypersensitive seedlings without green open cotyledons were selected and grown on soil for DNA isolation and progeny. For mapping, PCR-based markers such as simple-sequence-length polymorphism and cleaved-amplified polymorphic sequence markers were used (Supplemental Table III). Isolation of genomic DNA and PCR conditions have been described previously (Hirayama et al., 1999).

In Vitro PP2C Assay

cDNA fragments of AHG3 and the ahg3-1 mutant were generated by RT-PCR using primers AHG3FSmaI and AHG3RSalI (Supplemental Table IV), and sequenced. The cDNA fragment of the ahg3^G145D mutant was generated by RT-PCR using primers G145DFBspHI and AHG3RSalI (Supplemental Table IV). The PCR product of ahg3^G145D was digested with BspHI/SalI and substituted for the NcoI/SalI fragment of the AHG3 cDNA. The cDNAs were inserted into the SmaI/XhoI site of pGEX6P-1. Recombinant proteins (GST-AHG3, GST-ahg3, and GST-ahg3^G145D) were expressed in Escherichia coli DH10b cells and affinity purified on glutathione Sepharose 4B resin (Bertauche et al., 1996). Protein phosphatase activity was determined on ³²P-labeled casein as substrate (Bertauche et al., 1996; Gosti et al., 1999; Mackintosh and Moorhead, 1999; Merlot et al., 2001).

RNA Gel-Blot Analysis

Approximately 10 µg of total RNA were separated in 1.0% agarose gel containing 1.8% formaldehyde and transferred to a nylon membrane. The blot was hybridized at 60°C overnight in Church buffer (7% SDS, 0.5 M sodium phosphate buffer, pH 7.2, 10 mM EDTA), and then washed twice with 1x SSC, 0.1% SDS for 15 min, and twice with 0.1x SSC, 0.1% SDS for 20 min at 60°C. BAStation (Fuji Film) was used for visualization of the blot.

Transgenic Plants

The AHG3 and ahg3^G145D cDNAs were inserted into the SmaI/XhoI site of binary vector pMSH2. The At3g11410 (AHG3) genomic DNA segment was amplified by PCR using primers AHG3FSalI and AHG3RSmaI (Supplemental Table IV) and sequenced. For the ahg3^G145D mutation, the genomic fragment was generated by PCR using primers AHG3F3 and G145DRBspHI (Supplemental Table IV), digested with NcoI/BspHI, and substituted for the NcoI fragment of the genomic AHG3 clone. Cloned fragments were inserted into binary vector pBI101. Agrobacterium GV3101 cells were transformed with these plasmids and used for infection of flowering plants by the floral-dip method (Clough and Bent, 1998).

T-DNA Insertion Lines

T-DNA insertion lines were obtained from the Arabidopsis Biological Resource Center (ABRC). Homozygous plants were identified by the kanamycin tolerance test and a PCR-based method using T-DNA left- or right-border primers (Alonso et al., 2003) and gene-specific primers.

Generation of Double Mutants

ahg3-1 plants were crossed with abi1-1, abi2-1, abi3-1, abi4-1, and abi5-1 plants, and resulting F₁ plants were allowed to self pollinate. F₂ plants carrying both mutations were identified by mutant-specific cleaved-amplified polymorphic sequence markers (Supplemental Table I).

Chlorophyll Measurement

Chlorophyll was extracted from 10-d-old plantlets in 80% acetone. Chlorophyll content was determined as described previously (Arnon, 1949).

Measurement of Endogenous ABA Content

Samples were homogenized and extracted in 5 mL of methanol:water:acetic acid (90:9:1, v/v/v) with 2,6-di-tert-butyl-4-methylphenol (200 mg/L). ¹³C₂-ABA was added as an internal standard at the beginning of each extraction (Asami et al., 1999). After 17.5 mL of water were added, the supernatants were clarified by centrifugation and were loaded onto Oasis HLB cartridges (Waters). ABA was eluted with 1 mL methanol:water:acetic acid (90:9:1, v/v/v). An aliquot (5 mL) of each sample was loaded onto a Capcell Pac C₁₈ column (150 mm x 2 mm; Shiseido), and HPLC was performed using a binary solvent system of methanol and water (1:1, v/v) with 0.1% formic acid at a flow rate of 0.2 mL/min. The compounds were analyzed by tandem mass spectrometry with multiple reaction monitoring in negative-ion mode. The precursor ion (m/z) and product ion (m/z) set for each compound were as follows: 263 and 153 for the ABA standard and 265 and 153 for the ¹³C₂-ABA internal standard.

	ACKNOWLEDGMENTS

 
We thank Dr. R. Yoshida for helpful discussions and Dr. J. Schroeder and Dr. J. Kuhn for sharing their unpublished results and fruitful discussions. We are grateful to the Arabidopsis Biological Resource Center for providing various Arabidopsis lines.

Received August 24, 2005; returned for revision October 14, 2005; accepted October 23, 2005.

	FOOTNOTES

 
¹ This work was supported in part by a Grant-in-Aid from the Ministry of Education, Sports, Culture, Science and Technology of Japan and The Institute of Physical and Chemical Research President's Special Research Grant (to T.H.) and in part by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences (to K.S.).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Takashi Hirayama (hirayama{at}gsc.riken.jp).

^[W] The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.070128.

^* Corresponding author; e-mail hirayama{at}gsc.riken.jp; fax 81–45–508–7363.

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