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First published online December 22, 2006; 10.1104/pp.106.084103 Plant Physiology 143:745-758 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Ectopic Expression of ABSCISIC ACID 2/GLUCOSE INSENSITIVE 1 in Arabidopsis Promotes Seed Dormancy and Stress Tolerance1,[C],[OA]Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, Republic of China (P.-C.L, S.-G.H., W.-H.C.); and Department of Biological Science, Tokyo Metropolitan University, Hachioji-shi, Tokyo 192–0397, Japan (A.E., M.O., T.K.)
Abscisic acid (ABA) is an important phytohormone that plays a critical role in seed development, dormancy, and stress tolerance. 9-cis-Epoxycarotenoid dioxygenase is the key enzyme controlling ABA biosynthesis and stress tolerance. In this study, we investigated the effect of ectopic expression of another ABA biosynthesis gene, ABA2 (or GLUCOSE INSENSITIVE 1 [GIN1]) encoding a short-chain dehydrogenase/reductase in Arabidopsis (Arabidopsis thaliana). We show that ABA2-overexpressing transgenic plants with elevated ABA levels exhibited seed germination delay and more tolerance to salinity than wild type when grown on agar plates and/or in soil. However, the germination delay was abolished in transgenic plants showing ABA levels over 2-fold higher than that of wild type grown on 250 mM NaCl. The data suggest that there are distinct mechanisms underlying ABA-mediated inhibition of seed germination under diverse stress. The ABA-deficient mutant aba2, with a shorter primary root, can be restored to normal root growth by exogenous application of ABA, whereas transgenic plants overexpressing ABA2 showed normal root growth. The data reflect that the basal levels of ABA are essential for maintaining normal primary root elongation. Furthermore, analysis of ABA2 promoter activity with ABA2::  -glucuronidase transgenic plants revealed that the promoter activity was enhanced by multiple prolonged stresses, such as drought, salinity, cold, and flooding, but not by short-term stress treatments. Coincidently, prolonged drought stress treatment led to the up-regulation of ABA biosynthetic and sugar-related genes. Thus, the data support ABA2 as a late expression gene that might have a fine-tuning function in mediating ABA biosynthesis through primary metabolic changes in response to stress.
Plant growth and development are well regulated by the integration of external environmental cues and internal signals. The latter are involved in the production and action of phytohormones. Of these, abscisic acid (ABA) plays an important role in controlling many aspects of plant growth and development. For instance, during seed development and maturation, ABA stimulates the accumulation of protein and lipid storage reserves (Finkelstein and Somerville, 1988  ; Rock and Quatrano, 1995) and desiccation tolerance and dormancy (Karssen et al., 1983; Koornneef et al., 1989). It also mediates stomatal closure (Leung and Giraudat, 1998) to control water loss or transpiration rate. Endogenous ABA levels may regulate vegetative growth (for review, see Finkelstein et al., 2002) and help plants tolerate environmental stresses (drought, salt, and, to a lesser extent, low temperature; Leung and Giraudat, 1998; Qin and Zeevaart, 2002; Xiong et al., 2002). Recent studies also reveal that ABA may alter plant susceptibility to pathogen infection (Audenaert et al., 2002) and inhibit flowering (Razem et al., 2006).
Despite their physiological significance, expression and regulation of ABA biosynthetic genes at molecular levels were not well understood until recent years. Molecular genetic and biochemical studies have made great strides toward better understanding ABA biosynthesis and regulation in the past decade (for reviews, see Finkelstein et al., 2002
ABA biosynthesis in higher plants occurs through an indirect pathway (Schwartz et al., 2003
ABA has been considered a plant stress hormone because it is highly induced in vegetative tissues under stress conditions; the induction is associated with the up-regulation of ABA biosynthetic genes (such as ZEP, NCED, AAO3, and ABA3). However, these transcripts or ABA levels are reduced or abolished in most mutant alleles, which reflects the positive feedback regulatory circuit of ABA biosynthesis (for review, see Xiong and Zhu, 2003
Our previous data showed that ABA2 is a unique gene whose regulation is distinct from other ABA biosynthetic genes in some aspects (Cheng et al., 2002
Isolation and Characterization of ABA2 Overexpression Lines Most of the ABA-deficient mutants display a wilty phenotype and, to some extent, a small plant size relative to their wild type. To further demonstrate whether constitutive expression of ABA2 (At1g52340) may increase ABA2 expression and lead to larger plant size or other unexpected phenotypes, ABA2 overexpression transgenic plants were generated. The ABA2 transgene in transgenic plants was driven by a 35S promoter and contained the BAR gene for Basta herbicide resistance as a selectable marker. More than 10 independent transgenic lines were isolated on the basis of herbicide resistance. Three homozygous transgenic lines (4-3, 4-4, and 5-1) were randomly chosen for further study. Under normal growth conditions in soil, the overexpression lines exhibited no apparent phenotypic differences in aerial structures from the wild type, but the aba2 mutant displayed a typical mutant phenotype, with small plant size and early flowering (Fig. 1A ).
Southern-blot analysis further demonstrated that these three transgenic lines contained three or four copies of the T-DNA insertion (data not shown). Reverse transcription (RT)-PCR revealed that these lines have considerably higher levels of ABA2 transcripts than the wild type when grown in soil (Fig. 1B), in liquid culture, or on agar plates (data not shown). The ABA2 transcript shown in the aba2 mutant was slightly shorter than that of wild type and transgenic lines because of the 53-bp deletion in the mutant (Fig. 1B).
Because the level of ABA content controls seed dormancy, we tested whether ABA2 overexpression lines might increase the ABA level and result in deeper seed dormancy. As shown in Figure 2 , germination of the three transgenic lines was delayed relative to that of the wild type without cold pretreatment. For instance, after 2 d, wild type had 93.7% ± 3.5% seed germination, whereas transgenic lines 4-3, 4-4, and 5-1 had only 55.4% ± 8%, 51.8% ± 2.7%, and 36.5% ± 2.3% germination, respectively (Fig. 2A). With cold pretreatment for 3 d at 4°C, the germination delay for transgenic lines was impaired after 2-d growth on agar plates with 1% Suc (Fig. 2B).
A similar seed germination delay in overexpression lines was also shown on 2% and, to a lesser extent, on 6% Glc agar plates (Fig. 2, C and D). As expected, the ABA-deficient mutant aba2 always germinated earlier than the wild type and the overexpression lines throughout the germination period tested. The data suggest that the delayed germination pattern shown in the overexpression lines is reduced at the higher sugar concentration. Compared to seeds grown on 1% Suc, those grown on 2% Glc had a lower germination rate. Low sugar concentration promotes, but high sugar concentration inhibits, seed germination (Zhou et al., 1998 ; Arenas-Huertero et al., 2000; Finkelstein and Lynch, 2000). Delayed germination was observed in all genotypes on 6% Glc as compared to 2% Glc. For instance, 6-d germination rates on 6% Glc for aba2, wild type, and transgenic overexpressing lines 4-3, 4-4, and 5-1 were 88.9% ± 5.9%, 32% ± 2.8%, 19.8% ± 4.6%, 18% ± 4.2%, and 24.3% ± 7.4%, respectively; however, germination rates over 90% were observed in all genotypes on 2% Glc. It is noteworthy that wild type and overexpression lines showed seed germination followed by developmental arrest on 6% Glc (Fig. 2E). This observation can be explained by Glc-induced ABA accumulation that further suppresses seedling growth and development. However, the ABA-deficient mutant aba2 lacks such inhibition and grows steadily and develops true leaves (Cheng et al., 2002). This notion was further confirmed by the addition of fluridone, an ABA biosynthesis inhibitor, to the medium; wild type and ABA2 overexpression lines showed an aba2 phenocopy, with root elongation, cotyledon expansion, and true leaf development on fluridone-containing medium (Fig. 2E). Thus, the developmental arrest displayed on 6% Glc is most likely due to de novo ABA biosynthesis and accumulation. Fluridone is a selective inhibitor of carotenoid synthesis, which interferes with desaturation steps of carotenoid biogenesis and further blocks the accumulation of carotenoids. Thus, fluridone treatment inhibits carotenoid biosynthesis and leads to an albino-like seedling phenotype due to photooxidation of chlorophyll (Henson, 1984).
Although ABA2 transgenic plants showed no visible differences from wild type at aerial parts (Fig. 1A), our previous data showed strong ABA2 expression in root tissue (Cheng et al., 2002
Effect of Osmoticum on Seedling Growth and Development
It has been reported that overexpression of NCED3 causes elevated levels of ABA and enhances plant tolerance to drought in Arabidopsis (Iuchi et al., 2001
It is surprising that the aba2 mutant had the lowest damage (11.7% ± 3.8%) under the same growth conditions. Similar results were also observed in another ABA-deficient mutant, sto1/nced3, versus its corresponding wild type (C24; data not shown). The reason for this discrepancy remains to be illustrated. One of the possibilities might be related to an increase in stomatal aperture under such conditions. Ruggiero et al. (2004) reported that the sto1/nced3 mutant's stomatal aperture at day and night is larger than that of wild type under high humidity conditions. This finding might explain why the aba2 mutant had a higher survival rate because of its efficient absorption of water vapor from the air within the high-humidity petri dish. We also observed that wild-type and transgenic plants showed anthocyanin accumulation in newly growing true leaves with a slower growth rate, whereas the aba2 mutant retained light greening with a higher growth rate or had a larger plant size on average under low-water-potential conditions (Fig. 4A).
ABA has long been believed to be a stress-related phytohormone whose biosynthesis or level can be induced by various stresses. To further determine whether the seed germination delay shown on sugar medium also occurs under salinity stress, we grew seeds on 1% Suc agar plates with or without NaCl. As shown with sugar treatments, the aba2 mutant showed early germination regardless of the presence or absence of NaCl (compare Figs. 2 and 5 ). For instance, the aba2 mutant began germination within 24 h with 125 mM NaCl and 48 h with 250 mM NaCl, whereas germination of wild type and transgenic lines was delayed until 24 h with 125 mM NaCl (Fig. 5A) and 72 h with 250 mM NaCl (Fig. 5B). The higher NaCl concentration delayed seed germination in all genotypes. Transgenic plants showed a slight germination delay relative to wild type at 125 mM NaCl (Fig. 5A), whereas transgenic lines germinated in a manner similar to the wild type at 250 mM NaCl (Fig. 5B). At 125 mM NaCl, germination rates within the first 2 d for aba2, wild type, 4-3, 4-4, and 5-1 were 90.8% ± 1.8%, 76.8% ± 3.9%, 65.6% ± 8.1%, 57.7% ± 6.8%, and 54.2% ± 2.4%, respectively. At 250 mM NaCl, the aba2 mutant displayed expanded cotyledons with light greening, but no true leaf development after 7 d. In contrast, wild type and overexpression lines germinated and then showed developmental arrest (Fig. 5C). The light greening cotyledons in aba2 became bleached after 18 d of germination; the bleached seedlings were not viable after being transferred to fresh 1% Suc medium without NaCl, whereas the arrested seedlings of wild type and overexpression lines restored their green cotyledons and normal true leaf growth thereafter (data not shown). This observation indicates that wild type and overexpression lines have a self-protecting ability modulated by ABA and inhibited further growth and development under high NaCl conditions. NaCl-induced developmental arrest can be overcome with 1 µM fluridone: Wild type and overexpression lines showed the aba2 phenocopy with expanded cotyledons (Fig. 5C). The result indicates that NaCl-induced developmental arrest is most likely due to de novo ABA biosynthesis and accumulation.
Furthermore, immunoassays were used to determine ABA levels in seedlings from each genotype treated with 125 and 250 mM NaCl, respectively. The results exhibited that the aba2 mutant had only 45.3% (Fig. 5D) and 45.7% (Fig. 5E) of the wild-type ABA levels at 125 and 250 mM NaCl, respectively. However, transgenic lines were, on average, approximately 1.74- and 2.11-fold (Fig. 5, D and E, respectively) higher in ABA levels than that of wild type at 125 and 250 mM NaCl, respectively. The average increase of ABA level in these three transgenic lines is statistically significant, with P < 0.05 (P = 0.024 at 125 mM NaCl and P = 0.025 at 250 mM NaCl), Student's t test. Surprisingly, transgenic seedlings with high ABA levels did not show a germination delay when grown on 250 mM NaCl. Thus, the data suggest that there are distinct mechanisms that may impair or overcome ABA-mediated inhibition of seed germination under severe salinity stress. Phenotypic comparison revealed that the aba2 seedlings at 200 mM NaCl had lighter greening and glassier leaves than wild type and overexpression line seedlings (Fig. 5F). When grown on 1% Suc agar plates containing various NaCl concentrations (125–200 mM), seeds may germinate and develop true leaves; upon prolonged culture, however, the seedlings became bleached and died. As shown in Figure 5, F and G, although the aba2 mutant revealed early germination on NaCl-containing medium, more aba2 bleached seedlings were observed after prolonged culture. For instance, with 200 mM NaCl, wild type, aba2, and ABA2 overexpression lines had bleached seedling frequencies of 55.8% ± 3%, 81.6% ± 5.7%, and 39.3% ± 9.6%, respectively. In soil, after 21-d-old seedlings were watered with increasing concentrations of NaCl (50, 100, 150, and 200 mM NaCl), each for 4 d for a total of 16 d, the aba2 mutant became bleached (Fig. 5H). However, the wild type had just begun to bleach and the transgenic line had only dark greening rosette leaves with anthocyanin accumulation. These results indicate that ABA2 overexpression lines were more tolerant to salinity than wild type when they were grown on agar plates or in soil. Moreover, tolerance to salinity is associated with elevated ABA levels in transgenic lines.
Our previous data showed that ABA2 is a unique gene because its regulation is distinct from other ABA biosynthetic genes in response to stresses. For instance, ABA1, NCED3, and AAO3 transcripts are rapidly induced by dehydration and ABA treatment for 3 h, but ABA2 displays no conceivable change (Cheng et al., 2002 To determine ABA2 promoter activity under rapid dehydration, aerial parts of tissues from transgenic plants grown in soil for 21 d were removed and placed in an electronic dry box with 40% relative humidity for 1.5 and 3 h. As shown in Figure 6A , 1.5-h dehydration only slightly reduced ABA2 promoter activity as compared to control 1.5 h. These reductions were relatively small, about 28%, 38%, 19%, and 43% of the control activity for lines 1-11 (wild-type background), 4-12 (wild-type background), 3-6 (aba2 background), and 3-12 (aba2 background), respectively. However, 3-h dehydration showed no significant change of ABA2 promoter activity in each line as compared to that of the 3-h control. ABA2 promoter activity was considerably higher after 4 d of withholding water than that of the control; for instance, the transgenic line 1-11 (wild-type background) under 4-d drought had 16-fold higher activity than that of the control; strikingly, activity in the transgenic plant line 3-12 (aba2 background) under 4-d drought was 38.6-fold higher than that of the control. Three-day cold treatment also induced promoter activity higher than that of the control, but in transgenic lines 1-11 (wild-type background) and 3-6 (aba2 background) the activity was reduced by approximately 60% and 26%, respectively, as compared to that of 4-d drought. Although the control samples shown here (Fig. 6B) were harvested at 21 days after planting (dap), at the beginning of stress treatments, we also harvested the control samples at the same time points as the stress-treated samples. The results turned out to have very small variation among those control samples. For example, the control samples at day 1 (22 dap), day 3 (24 dap), and day 4 (25 dap) had, respectively, GUS activity change all below 0.49-, 0.43-, and 1.0-fold for every transgenic line tested relative to that of the control samples at day 0 (21 dap; data not shown). Because these variations were very small compared to the variations between stress-treated and control samples at day 0 (Fig. 6B), we believe that there was no significant developmental effect on ABA2 promoter activity among these control samples during this 4-d development (i.e. 21–25 dap), at least in this case.
For NaCl treatments, transgenic plants grown in soil for 3 weeks were soaked with water, 125 or 250 mM NaCl for 24 h; water was sufficient to induce ABA2 promoter activity in transgenic plants (both wild-type and aba2 background), albeit with less efficiency than with NaCl and drought treatment (Fig. 6B). As water flooding may cause a submergence or hypoxia effect, we observed that the ABA2 promoter was submergence inducible (data not shown). Transgenic plants (wild-type background) with NaCl treatment had a relative induction of ABA2 promoter activity with a value higher than with cold (4°C) treatment, but slightly lower than with 4-d drought treatment. After 14 d on agar plates, ABA2 promoter activity in both the wild-type and aba2 backgrounds was induced by NaCl treatment. Such induction was more pronounced with the addition of 125 mM NaCl in transgenic plants with aba2 background (Fig. 6C). As compared with 1% Suc treatment, the addition of NaCl increased promoter activity 3.2-fold at 125 mM and 6.9-fold at 250 mM NaCl in line 1-11 (wild-type background); activity in line 3-12 (aba2 background) was increased 11-fold at 125 mM NaCl but was alleviated to only 6.3-fold at 250 mM NaCl. Such reductions in lines 3-6 and 3-12 (aba2 background) are most likely due to low metabolism at 250 mM NaCl treatment for 14 d. At that time point, some transgenic plants in the mutant background started showing a bleached phenotype, whereas transgenic lines in the wild-type background remained with developmental arrest. Taken together, these findings indicate that ABA2 promoter activity was up-regulated by NaCl stress. In general, ABA2 promoter activity is remarkably induced by prolonged cold, drought, flooding, and salinity, but not short-term dehydration. Induction is profound in transgenic lines with an aba2 background. It indicates that the feedback inhibition of ABA2 promoter activity by ABA is likely missing in the ABA-deficient mutant aba2. Thus, mutants may build up considerable levels of ABA2 promoter activity relative to transgenic lines under a wild-type background.
As mentioned above, because ABA2 promoter activity was considerably changed under prolonged stress conditions but not short rapid stress treatment, ABA2 expression is controlled differently from other ABA biosynthetic genes that show early stress response. One of the possibilities is likely due to the primary metabolic change during prolonged stress treatments, which in turn regulate ABA2 expression. Because sugars play a central role in plant growth and development and ABA biosynthetic genes are up-regulated by sugars, we tested for alterations in sugar-related gene expression under prolonged stress conditions at the molecular level. As shown in Figure 7 , the ABA2 transcript was overexpressed in transgenic plants grown in soil for 3 weeks followed by drought treatment for 4 d or without drought treatment. The levels of the ABA2 transcript were elevated in the wild type after 4-d drought treatment, in agreement with the induction of ABA2 promoter activity (Fig. 6B). However, because the aba2 transcript has a 53-bp deletion, its stability might be labile so that the accumulation of the aba2 transcript was not as high as its promoter activity in response to 4-d drought. Similarly, the NCED3 (At3g14440) transcript was also induced under the same growth conditions. It is interesting that the aba2 mutant accumulated a higher amount of NCED3 transcript than that of wild-type and transgenic plants. The reason for this accumulation remains unknown. One of the possibilities is that NCED3 expression is inducible by ABA and drought may increase ABA biosynthesis and accumulation; however, the aba2 mutant lacks ABA under drought conditions and might also result in a loss of negative feedback regulation of NCED3 expression.
For sugar-related genes, AtHXK1 (At4g29130), a sugar sensor, and AtSusy1 (At5g20830), a Suc-cleaving enzyme, were both up-regulated under stress conditions, whereas AtSusy2 (At5g49190) showed low to undetectable levels (data not shown). The up-regulation patterns of AtHXK1 and AtSusy1 expression in ABA2 transgenic plants under drought conditions were impaired or unchanged as compared to the wild type and aba2 mutant. Thus, these results provide evidence that late expression of ABA2 in response to drought, at least in part, is associated with up-regulated expression of sugar-related genes.
Overexpression of ABA2 in Arabidopsis Increases ABA Content and Promotes Seed Germination Delay and NaCl Tolerance
It has been reported that overexpression of ABA biosynthetic genes results in an elevated ABA level and extended seed dormancy under nonstress conditions (Frey et al., 1999
It is believed that plants have multiple stress perceptions and signal transduction pathways to generate specific and common responses to stresses. Although the common response may have cross talk at various steps in the pathways, the specific response may reflect the unique pathway induced only by specific stresses (for review, see Chinnusamy et al., 2004
In addition to its function as a stress hormone, ABA has long been considered a plant growth inhibitor because the elevated endogenous ABA content induced by stress or exogenous ABA application normally suppresses vegetative tissue growth. However, most ABA-deficient mutants have a smaller size than the wild type, which reflects that the basal level of ABA is essential for normal plant growth and development. It is interesting that all overexpression lines of ABA biosynthetic genes reported to date, including those in this study, show a phenotype not different from that of wild type in aerial parts under normal growth conditions. However, because ABA2 is a unique gene, its regulation and response to stresses are different from other ABA biosynthetic genes in some aspects. In addition, ABA2 tissue-specific expression is predominantly restricted to vascular bundles in many tissues, including roots. The latter compelled us to examine the effect of ABA on root growth.
Our data demonstrate that the aba2 mutant had a shorter primary root length than that of wild-type and transgenic plants (Fig. 3, A and B). The reduction is primarily due to ABA deficiency because exogenous application of ABA into the medium restored the root growth in the mutant to normal. We observed that ABA sensitivity differs in distinct ecotypes, to some extent, but the short primary root length caused by the lack of ABA was ecotype independent (Fig. 3, B and D). It is noteworthy that primary root elongation was not inhibited by low amounts of exogenous ABA (less than 500 nM); with more than 500 nM ABA, plants normally show retarded growth and have inhibited primary root elongation and decreased lateral root numbers. ABA inhibits radicle emergence, but not seedling growth; however, such an inhibitory effect can be suppressed with the presence of low concentrations of sugar (Finkelstein and Lynch, 2000
Our previous data demonstrated that ABA2 is a unique gene whose regulation is different from other ABA biosynthetic genes, such as AtABA1, AtNCED3, AtABA3, and AAO3, in response to stresses. The latter genes are transcriptionally up-regulated under short-term drought conditions (
The reason for the discrepant regulation between ABA2 and other ABA biosynthetic genes is unknown. Analysis of the ABA2 promoter within 2.8 kb upstream of the translational start site (ATG) using the Arabidopsis Gene Regulatory Information Server (agris; Ohio State University; http://Arabidopsis.med.ohio-state.edu/AtcisDB) and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare; Lescot et al., 2002
Plant Materials and Growth Conditions
Plant materials used in this study were Arabidopsis (Arabidopsis thaliana) ecotype Columbia. The aba2 mutant is a gin1-3 allele with a 53-bp deletion at the start of the second exon (Cheng et al., 2002
Unless stated otherwise, seeds in this study were sterilized and subjected to cold pretreatment at 4°C for 3 d in the dark, and then grown on agar plates or in soil as the first day of seed germination or planting. Seed germination conditions were at 24°C under long-day conditions with a 16-h-light/8-h-dark cycle with light intensity approximately 80 µE s–1 m–2. For aseptic growth, seeds were sterilized with 70% ethanol for 1 min and one-half-strength commercial bleach for 12 min, followed by five washes with sterilized water, and placed at 4°C for 3 d in the dark for cold treatment. Subsequently, seeds were transferred to modified Murashige and Skoog (Murashige and Skoog, 1962
For overexpression, ABA2 (GIN1) full-length cDNA was amplified by RT-PCR and cloned into pGEM-T Easy vector (Promega), followed by subcloning into the binary vector (pSMAB704) driven by a constitutive 35S promoter (35S::ABA2). For tissue-specific expression and GUS assay, the ABA2 promoter, approximately 2.9 kb upstream of the ATG start codon, was amplified by PCR and fused to a GUS coding region to generate ABA2::GUS in the pSMAB704 binary vector. Transgene constructs were confirmed by sequencing and subsequently transformed into wild type or the aba2 mutant (T0) by use of the floral-dip method (Clough and Bent, 1998
For germination tests, seeds harvested from the same batches were cold pretreated and then grown on modified Murashige and Skoog medium supplemented with Glc, Suc, NaCl, ABA, glufosinate ammonium, or fluridone at various concentrations listed in the "Results." The medium was autoclaved and cooled to 50°C to 60°C prior to the addition of filter-sterilized ABA, glufosinate ammonium, or fluridone. For root elongation experiments, cold-pretreated seeds from different genotypes were first grown on agar plates with 1% Suc for 4 or 5 d, then uniform seedlings of similar size and primary root length were transferred to appropriate fresh medium and grown vertically for another 6 or 7 d.
One-half-strength modified Murashige and Skoog medium (pH 5.7) with 0.7% Phyto agar (Duchefa Biochemie B.V.) was autoclaved. The sterilized medium was cooled to 50°C to 60°C and then aliquoted into petri dishes (100- x 20-mm depth), 20 mL each, for solidification. PEG-infused plates were made by dissolving PEG-8000 (Sigma) powder into one-half-strength Murashige and Skoog solution (pH 5.7) with the above-mentioned components, except phyto agar, and then filter sterilized; this PEG solution was then overlaid on agar-solidified medium at a ratio of 3:2 (v/v) and equilibrated overnight (
Wild type, aba2, and ABA2 overexpression seeds with cold pretreatment were grown on agar plates with various NaCl concentrations for 18 or 28 d. The ratios of bleached to total plants were counted to define tolerance to salinity. Seeds with cold pretreatment were also grown in soil for 3 weeks; then plants were subjected to watering with solutions containing four gradually increased concentrations of NaCl (50, 100, 150, and 200 mM), each for 4 d for a total of 16 d (Shi et al., 2003
Cold-pretreated transgenic seeds harboring the ABA2::GUS transgene were grown on agar plates or in soil for different time periods, depending on experiments, and then subjected to various stresses (cold, drought, or salinity). The treated plants were harvested and ground with extraction buffer (50 mM NaHPO4 [pH 7.0], 10 mM
Total RNA was extracted from wild type, aba2, and ABA2 overexpression lines using TRIzol reagent (Invitrogen). Six micrograms of total RNA of each genotype with 1 µg oligo(dT) primer (Invitrogen) were heated at 70°C for 5 min and then chilled on ice immediately. RNA was then subjected to RT with reverse-transcriptase Avian myeloblastosis virus (Roche) at 42°C for 1 h according to the manufacturer's protocol. Synthesized cDNA was used as a template for PCR.
For ABA extraction, seedlings harvested at appropriate stages or after stress treatments were treated with extraction buffer (80% methanol and 2% glacial acetic acid) for 24 h under darkness, followed by centrifugation for 10 min at 2,000g. Supernatants were taken up and dried in a speedvac, then resuspended in 100% methanol plus 0.2 M NH4H2PO4 (pH 6.8) for 10 min. To avoid plant pigment and other nonpolar compound effects on the immunoassay, the extracts were first passed through a polyvinylpolypyrrolidone column and then C18 cartridges. Elutes were concentrated to dryness in a speedvac and resuspended in Tris-buffered saline for immunoassay (Hsu and Kao, 2003
We thank Dr. Charles M. Papa (Tajen University, PingTung, Taiwan, Republic of China), Dr. Jen Sheen (Department of Molecular Biology, Massachusetts General Hospital, Boston), and Dr. Liming Xiong (Donald Danforth Plant Science Center, St. Louis) for critically reading this manuscript, and Dr. H. Ichikawa (Department of Plant Biotechnology, National Institute of Agrobiological Sciences, Tsukuba, Japan) for providing the binary vector pSMAB704. We are also grateful to Dr. C.H. Kao and Ms. Y.T. Hsu (Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China) for assistance with ABA immunoassay. Received May 25, 2006; accepted December 10, 2006; published December 22, 2006.
1 This work was supported by the National Science Council and Academia Sinica (grant nos. NSC 349C47B and AS 91IB1PP, respectively, to W.-H.C.), Taiwan, Republic of China. 
2 These authors contributed equally to the paper. 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: Wan-Hsing Cheng (whcheng{at}gate.sinica.edu.tw).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.084103 * Corresponding author; e-mail whcheng{at}gate.sinica.edu.tw; fax 886–2–2782–7954.
Arenas-Huertero F, Arroyo A, Zhou L, Sheen J, Leon P (2000) Analysis of Arabidopsis glucose insensitive mutants, gin5 and gin6, reveals a central role of the plant hormone ABA in the regulation of plant vegetative development by sugar. Genes Dev 14: 2085–2096 Audenaert K, De Meyer GB, Höfte MM (2002) Abscisic acid determines basal susceptibility of tomato to Botrytis cinerea and suppresses salicylic acid-dependent signaling mechanisms. Plant Physiol 128: 491–501 Audran C, Borel C, Frey A, Sotta B, Meyer C, Simonneau T, Marion-Poll A (1998) Expression studies of the zeaxanthin epoxidase gene in Nicotiana plumbaginifolia. Plant Physiol 118: 1021–1028 Bittner F, Oreb M, Mendel RR (2001) ABA3 is a molybdenum cofactor sulfurase required for activation of aldehyde oxidase and xanthine dehydrogenase in Arabidopsis thaliana. J Biol Chem 276: 40381–40384 Burbidge A, Grieve TM, Jackson A, Thompson A, McCarty DR, Taylor IB (1999) Characterization of the ABA-deficient tomato mutant notabillis and its relationship with maize Vp14. Plant J 17: 427–431[CrossRef][Web of Science][Medline] Cheng W-H, Endo A, Zhou L, Penney J, Chen H-C, Arroyo A, Leon P, Nambara E, Asami T, Seo M, et al (2002) A unique short-chain dehydrogenase/reductase in Arabidopsis glucose signaling and abscisic acid biosynthesis and functions. Plant Cell 14: 2723–2743 Chernys JT, Zeevaart JAD (2000) Characterization of the 9-cis-epoxycarotenoid dioxygenase gene family and the regulation of abscisic acid biosynthesis in avocado. Plant Physiol 124: 343–353 Chinnusamy V, Schumaker K, Zhu JK (2004) Molecular genetic perspectives on cross-talk and specificity in abiotic stress signaling in plants. J Exp Bot 55: 225–236 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743[CrossRef][Web of Science][Medline] Déjardin A, Sokolov LN, Kleczkowski LA (1999) Sugar/osmoticum levels modulate differential abscisic acid-independent expression of two stress-responsive sucrose synthase genes in Arabidopsis. Biochem J 344: 503–509[CrossRef][Web of Science][Medline] De Smet I, Signora L, Beeckman T, Inzé K, Foyer CH, Zhang H (2003) An abscisic acid-sensitive checkpoint in lateral root development of Arabidopsis. Plant J 33: 543–555[CrossRef][Web of Science][Medline] Finkelstein RR, Gampala SS, Rock CD (2002) Abscisic acid signaling in seeds and seedlings. Plant Cell 14: S15–S45 Finkelstein RR, Lynch TJ (2000) Abscisic acid inhibition of radicle emergence but not seedling growth is suppressed by sugars. Plant Physiol 122: 1179–1186 Finkelstein R, Somerville C (1988) Abscisic acid and high osmoticum promote accumulation of long-chain fatty acids in developing embryos of Brassica napus. Plant Sci 61: 213–217[CrossRef] Frey A, Audran C, Marin E, Sotta B, Marion-Poll A (1999) Engineering seed dormancy by the modification of zeaxanthin epoxidase gene expression. Plant Mol Biol 39: 1267–1274[CrossRef][Web of Science][Medline] Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50: 151–158[CrossRef][Web of Science][Medline] Gibson SI (2000) Plant sugar-response pathways: part of a complex regulatory web. Plant Physiol 124: 1532–1539 González-Guzmán M, Apostolova N, Bellés JM, Barrero JM, Piqueras P, Ponce MR, Micol JL, Serrano R, Rodríguez PL (2002) The short-chain alcohol dehydrogenase ABA2 catalyzes the conversion of xanthoxin to abscisic aldehyde. Plant Cell 14: 1833–1846 Henson EA (1984) Inhibition of abscisic acid accumulation in seedling shoots of Pearl Millet (Pennisetum americanum [L] Leeke) following induction of chlorosis by norflurazon. Z Pflanzenphysiol 114: 35–43 Hsu YT, Kao CH (2003) Role of abscisic acid in cadmium tolerance of rice (Oryza sativa L.) seedlings. Plant Cell Environ 26: 867–874[CrossRef][Medline] Iuchi S, Kobayashi M, Taji T, Naramoto M, Seki M, Kato T, Tabata S, Kakubari Y, Yamaguchi-Shinozaki K, Shinozaki K (2001) Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J 27: 325–333[CrossRef][Web of Science][Medline] Iuchi S, Kobayashi M, Yamaguchi-Shinozaki K, Shinozaki K (2000) A stress-inducible gene for 9-cis-epoxycarotenoid dioxygenase involved in abscisic acid biosynthesis under water stress in drought-tolerance cowpea. Plant Physiol 123: 553–562 Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: Karssen CM, Brinkhorst-van der Swan DLC, Breekland AE, Koornneef M (1983) Induction of dormancy during seed development by endogenous abscisic acid: studies of abscisic acid deficient genotypes of Arabidopsis thaliana (L.) Heynh. Planta 157: 158–165[CrossRef][Web of Science] Koiwai H, Nakaminami K, Seo M, Mitsuhashi W, Toyomasu T, Koshiba T (2004) Tissue-specific localization of an abscisic acid biosynthetic enzyme, AAO3, in Arabidopsis. Plant Physiol 134: 1697–1707 Koornneef M, Hanhart CJ, Hilhorst HWM, Karssen CM (1989) In vivo inhibition of seed development and reserve protein accumulation in recombinants of abscisic acid biosynthesis and responsiveness mutants in Arabidopsis thaliana. Plant Physiol 90: 463–469 Laby RJ, Kincaid MS, Kim D, Gibson SI (2000) The Arabidopsis sugar-insensitive mutants sis4 and sis5 are defective in abscisic acid synthesis and response. Plant J 23: 587–596[CrossRef][Web of Science][Medline] Léon-Kloosterziel KM, Alvarez-Gil M, Ruijs GJ, Jacobsen SE, Olszewski NE, Schwartz SH, Zeevaart JAD, Koornneef M (1996) Isolation and characterization of abscisic acid-deficient Arabidopsis mutants at two new loci. Plant J 10: 655–661[CrossRef][Web of Science][Medline] Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, de Peer YV, Rouzé P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30: 325–327 Leung J, Giraudat J (1998) Abscisic acid signal transduction. Annu Rev Plant Physiol Plant Mol Biol 49: 199–222[CrossRef][Web of Science][Medline] Marin E, Nussaume L, Quesada A, Gonneau M, Sotta B, Hugueney P, Frey A, Marion-Poll A (1996) Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana. EMBO J 15: 2331–2342[Web of Science][Medline] Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue culture. Physiol Plant 15: 473–497[CrossRef] Quesada V, Ponce MR, Micol JL (2000) Genetic analysis of salt-tolerant mutants in Arabidopsis thaliana. Genetics 154: 421–436 Qin X, Zeevaart JAD (1999) The 9-cis-epoxycarotenoid cleavage reaction is the key regulatory step of abscisic acid biosynthesis in water-stressed bean. Proc Natl Acad Sci USA 96: 15354–15361 Qin X, Zeevaart JAD (2002) Overexpression of a 9-cis-epoxycarotnoid dioxygenase gene in Nicotiana plumbaginifolia increases abscisic acid and phaseic acid levels and enhances drought tolerance. Plant Physiol 128: 544–551 Razem FA, EI-Kereamy A, Abrams SR, Hill RD (2006) The RNA-binding protein FCA is an abscisic acid receptor. Nature 439: 290–294[CrossRef][Medline] Rock CD, Quatrano RS (1995) The role of hormones during seed development. In PJ Davies, ed, Plant Hormones: Physiology, Biochemistry and Molecular Biology. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 671–697 Rolland F, Moore B, Sheen J (2002) Sugar sensing and signaling in plants. Plant Cell 14: S185–S205 Rook F, Corke F, Card R, Munz G, Smith C, Bevan MW (2001) Impaired Suc-induction mutants reveal the modulation of sugar-induced starch biosynthetic gene expression by abscisic acid signaling. Plant J 26: 421–433[CrossRef][Web of Science][Medline] Ruggiero B, Koiwa H, Manabe Y, Quist TM, Inan G, Saccardo F, Joly RJ, Hasegawa PM, Bressan RA, Maggio A (2004) Uncoupling the effects of abscisic acid on plant growth and water relations. Analysis of sto1/nced3, an abscisic mutant in Arabidopsis. Plant Physiol 136: 3134–3147 Schwartz SH, Qin X, Zeevaart JAD (2003) Elucidation of the indirect pathway of abscisic acid biosynthesis by mutants, genes, and enzymes. Plant Physiol 131: 1591–1601 Schwartz SH, Tan BC, Gage DA, Zeevaart JAD, McCarty DR (1997) Specific oxidative cleavage of carotenoid by VP14 of maize. Science 276: 1872–1874 Seo M, Koshiba T (2002) Complex regulation of ABA biosynthesis in plants. Trends Plant Sci 7: 41–48[CrossRef][Web of Science][Medline] Seo M, Peeters AJM, Koiwai H, Oritani T, Marion-Poll A, Zeevaart JAD, Koornneef M, Kamiya Y, Koshiba T (2000) The Arabidopsis aldehyde oxidase 3 (AAO3) gene product catalyzes the final step in abscisic acid biosynthesis in leaves. Proc Natl Acad Sci USA 97: 12908–12913 Sharp RE, Wu Y, Voetberg GS, Saab IN, LeNoble ME (1994) Confirmation that abscisic acid accumulation is required for maize primary root elongation at low water potentials. J Exp Bot 45: 1743–1751[Web of Science] Shi H, Lee B-H, Wu S-J, Zhu JK (2003) Overexpression of a plasma membrane Na+/H+ antiporter gene improves salt tolerance in Arabidopsis thaliana. Nat Biotechnol 21: 81–85[CrossRef][Web of Science][Medline] Tan B-C, Joseph LM, Deng W-T, Liu L, Li Q-B, Cline K, McCarty DR (2003) Molecular characterization of the Arabidopsis 9-cis-epoxycarotenoid dioxygenase gene family. Plant J 35: 44–56[CrossRef][Web of Science][Medline] Tan B-C, Schwartz SH, Zeevaart JAD, McCarty DR (1997) Genetic control of abscisic acid biosynthesis in maize. Proc Natl Acad Sci USA 94: 12235–12240 Thompson AJ, Jackson AC, Parker RA, Morpeth DR, Burbidge A, Taylor IB (2000a) Abscisic acid biosynthesis in tomato: regulation of zeaxanthin epoxidase and 9-cis-epoxycarotenoid dioxygenase mRNAs by light/dark cycles, water stress and abscisic acid. Plant Mol Biol 42: 833–845[CrossRef][Web of Science][Medline] Thompson AJ, Jackson AC, Symonds RC, Mulholland BJ, Dadswell AR, Blake PS, Burbidge A, Taylor IB (2000b) Ectopic expression of a tomato 9-cis-epoxycarotenoid dioxygenase gene causes over-production of abscisic acid. Plant J 23: 363–374[CrossRef][Web of Science][Medline] Verslues PE, Bray EA (2004) LWR1 and LWR2 are required for osmoregulation and osmotic adjustment in Arabidopsis. Plant Physiol 136: 2831–2842 Xiong L, Ishitani M, Lee H, Zhu JK (2001) The Arabidopsis LOS5/ABA3 locus encodes a molybdenum cofactor sulfurase and modulates cold and osmotic stress-responsive gene expression. Plant Cell 13: 2063–2083 Xiong L, Lee H, Ishitani M, Zhu JK (2002) Regulation of osmotic stress-responsive gene expression by the LOS6/ABA1 locus in Arabidopsis. J Biol Chem 277: 8588–8596 Xiong L, Zhu JK (2003) Regulation of abscisic acid biosynthesis. Plant Physiol 133: 29–36 Zhou L, Jang JC, Jones TL, Sheen J (1998) Glucose and ethylene signal transduction crosstalk revealed by an Arabidopsis glucose-insensitive mutant. Proc Natl Acad Sci USA 95: 10294–10299 This article has been cited by other articles:
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w, according to Verslues and Bray, 2004
3 h), whereas ABA2 is not. The current study further confirms that ABA2 promoter activity had little change after a short period of drought treatment (
12 h). The excess PEG solution was then removed. The procedure essentially followed the protocol of Verslues and Bray (2004)
