ectopic expression of abscisic acid 2/glucose … · et al., 2002) and inhibit ﬂowering (razem et...

Ectopic Expression of ABSCISIC ACID 2/GLUCOSEINSENSITIVE 1 in Arabidopsis Promotes SeedDormancy and Stress Tolerance1[C][OA]

Pei-Chi Lin2, San-Gwang Hwang2, Akira Endo, Masanori Okamoto, Tomokazu Koshiba,and Wan-Hsing Cheng*

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 toler-ance. 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-overexpressingtransgenic plants with elevated ABA levels exhibited seed germination delay and more tolerance to salinity than wild typewhen grown on agar plates and/or in soil. However, the germination delay was abolished in transgenic plants showing ABAlevels over 2-fold higher than that of wild type grown on 250 mM NaCl. The data suggest that there are distinct mechanismsunderlying ABA-mediated inhibition of seed germination under diverse stress. The ABA-deficient mutant aba2, with a shorterprimary root, can be restored to normal root growth by exogenous application of ABA, whereas transgenic plants overex-pressing ABA2 showed normal root growth. The data reflect that the basal levels of ABA are essential for maintaining normalprimary root elongation. Furthermore, analysis of ABA2 promoter activity with ABA2Tb-glucuronidase transgenic plantsrevealed 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 ABAbiosynthetic and sugar-related genes. Thus, the data support ABA2 as a late expression gene that might have a fine-tuningfunction in mediating ABA biosynthesis through primary metabolic changes in response to stress.

Plant growth and development are well regulatedby the integration of external environmental cues andinternal signals. The latter are involved in the produc-tion and action of phytohormones. Of these, abscisicacid (ABA) plays an important role in controllingmany aspects of plant growth and development. Forinstance, during seed development and maturation,ABA stimulates the accumulation of protein and lipidstorage reserves (Finkelstein and Somerville, 1988;Rock and Quatrano, 1995) and desiccation toleranceand dormancy (Karssen et al., 1983; Koornneef et al.,1989). It also mediates stomatal closure (Leung andGiraudat, 1998) to control water loss or transpiration

rate. Endogenous ABA levels may regulate vegetativegrowth (for review, see Finkelstein et al., 2002) andhelp plants tolerate environmental stresses (drought,salt, and, to a lesser extent, low temperature; Leung andGiraudat, 1998; Qin and Zeevaart, 2002; Xiong et al.,2002). Recent studies also reveal that ABA may alterplant susceptibility to pathogen infection (Audenaertet al., 2002) and inhibit flowering (Razem et al., 2006).

Despite their physiological significance, expressionand regulation of ABA biosynthetic genes at molecularlevels were not well understood until recent years.Molecular genetic and biochemical studies have madegreat strides toward better understanding ABA bio-synthesis and regulation in the past decade (for re-views, see Finkelstein et al., 2002; Seo and Koshiba,2002; Schwartz et al., 2003; Xiong and Zhu, 2003). Thecombination of genetic and biochemical screens, espe-cially in Arabidopsis (Arabidopsis thaliana), identifiedseveral mutants in the ABA biosynthetic pathway(for review, see Gibson, 2000; Finkelstein et al., 2002;Rolland et al., 2002; Xiong and Zhu, 2003). Most of theseloci leading to mutant phenotypes have been clonedand characterized. For instance, AtABA1 (AtLOS6; ho-mologous to tobacco [Nicotiana tabacum] ABA2) en-codes a zeaxanthin epoxidase (ZEP) that catalyzesthe epoxidation of zeaxanthin and antheraxanthin toviolaxanthin in plastids (Marin et al., 1996; Xiong et al.,2002). After structural modification, violaxanthin is

1 This work was supported by the National Science Council andAcademia Sinica (grant nos. NSC 349C47B and AS 91IB1PP, respec-tively, to W.-H.C.), Taiwan, Republic of China.

2 These authors contributed equally to the paper.* Corresponding author; e-mail [email protected]; fax

886–2–2782–7954.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Wan-Hsing Cheng ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[OA] Open Access articles can be viewed online without a sub-scription.

www.plantphysiol.org/cgi/doi/10.1104/pp.106.084103

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converted to 9-cis-epoxycarotenoid. The epoxycarote-noids 9#-cis-neoxanthin and/or 9-cis-violaxanthin arethen oxidized by 9-cis-epoxycarotenoid dioxygenase(NCED) to generate a C15 intermediate, xanthoxin(Schwartz et al., 1997). The product xanthoxin is subse-quently transported to the cytosol and further convertedto abscisic aldehyde by a short-chain dehydrogenase/reductase 1 encoded by ABA2 in Arabidopsis (Leon-Kloosterziel et al., 1996; Rook et al., 2001; Cheng et al.,2002; Gonzalez-Guzman et al., 2002). Arabidopsis al-dehyde oxidase 3 (AAO3) catalyzes the last step in theconversion of abscisic aldehyde to ABA (Seo et al., 2000)and needs a molybdenum cofactor sulfurase encodedby AtABA3 (AtLOS5) for its activity (Leon-Kloosterzielet al., 1996; Bittner et al., 2001; Xiong et al., 2001).

ABA biosynthesis in higher plants occurs throughan indirect pathway (Schwartz et al., 2003), which usesthe NCED-cleaved neoxanthin (C40) product, xan-thoxin (C15), as the ABA biosynthetic precursor. Inaddition, NCED expression in response to environ-mental stresses is so rapid and dramatic that it hasbeen considered a key enzyme in the ABA biosyntheticpathway. As such, NCED has been studied extensivelyand identified in many plant species, such as maize(Zea mays; Tan et al., 1997), tomato (Lycopersiconesculentum; Burbidge et al., 1999), bean (Phaseolusvulgaris; Qin and Zeevaart, 1999), avocado (Persicaamericana; Chernys and Zeevaart, 2000), cowpea (Vignaunguiculata; Iuchi et al., 2000), and Arabidopsis (Iuchiet al., 2001). More recently, detailed study of tissue-specific expression of the AtNCED gene family revealeddifferential expression of AtNCED members in differ-ent tissues and subcellular localization in differentplastid compartments (Tan et al., 2003). The differen-tial membrane-binding capacity of AtNCEDs furtherimplies posttranslational regulation of NCED activity.It is surprising that the spatial and temporal expres-sion of ABA2 is primarily restricted to vascular bun-dles in roots, hypocotyls, and aged leaves, but not inABA target sites, such as guard cells or seeds (Chenget al., 2002). Further localization of AAO3, which ca-talyzes the last step of ABA biosynthesis and can beconsidered the ABA production site, also shows theprotein to be present in vascular bundles in roots,hypocotyls and inflorescence stems, and leaf veins;additionally, AAO3 is detectable in guard cells (Koiwaiet al., 2004). Taken together, these data suggest thedynamic mobility of ABA precursors and/or ABA tothe target sites despite the guard cells themselvesbeing capable of synthesizing ABA.

ABA has been considered a plant stress hormonebecause it is highly induced in vegetative tissues un-der stress conditions; the induction is associated withthe up-regulation of ABA biosynthetic genes (such asZEP, NCED, AAO3, and ABA3). However, these tran-scripts or ABA levels are reduced or abolished in mostmutant alleles, which reflects the positive feedbackregulatory circuit of ABA biosynthesis (for review,see Xiong and Zhu, 2003). Furthermore, tobacco ZEP(Audran et al., 1998) and tomato ZEP and NCED

(Thompson et al., 2000a) show a diurnal expressionpattern. Overexpression of NpZEP (Frey et al., 1999),tomato LeNCED1, and bean PvNCED1 in tobacco(Thompson et al., 2000b; Qin and Zeevaart, 2002)and AtNCED3 in Arabidopsis (Iuchi et al., 2001) dis-plays an increased ABA level in seeds and extendedseed dormancy. Transgenic tobacco plants expressingPvNCED1 driven by 35S or a dexamethasone-induciblepromoter and transgenic Arabidopsis plants over-expressing AtNCED3 exhibit drought tolerance (Iuchiet al., 2001; Qin and Zeevaart, 2002). A similar resultfor increased seed dormancy is also obtained by theoverexpression of ABSCISIC ACID INSENSITIVE (ABI)genes in Arabidopsis (for review, see Finkelstein et al.,2002). These data show an alternative way to gener-ate deeper seed dormancy and stress-tolerant plantsthrough genetic manipulation of ABA biosyntheticand/or responsive gene expression.

Our previous data showed that ABA2 is a uniquegene whose regulation is distinct from other ABA bio-synthetic genes in some aspects (Cheng et al., 2002).Most of the ABA biosynthetic genes, such as ABA1,NCED3, AAO3, and ABA3 in Arabidopsis, are up-regulated under stressful environments, whereas ABA2shows no obvious induction under short-term droughtor ABA treatment (Cheng et al., 2002; Gonzalez-Guzmanet al., 2002). It also remains unknown whether trans-genic plants overexpressing ABA2 show increasedABA levels, extended seed dormancy, and stress tol-erance. In this study, we provide genetic evidence thatoverexpressing ABA2 in Arabidopsis transgenic plantsleads to seed germination delay, maintenance of pri-mary root growth, and an elevated level of ABA.Results from this study also demonstrate that ABA2transgenic plants exhibit increased salinity tolerancerelative to wild type. Additionally, the increase of ABA2promoter activity and up-regulation of ABA2 and sugar-related genes under prolonged stress treatments fur-ther suggest that ABA2 is a late stress-responsive genewith a fine-tuning function in controlling ABA bio-synthesis in response to stress.

RESULTS

Isolation and Characterization of ABA2Overexpression Lines

Most of the ABA-deficient mutants display a wiltyphenotype and, to some extent, a small plant sizerelative to their wild type. To further demonstratewhether constitutive expression of ABA2 (At1g52340)may increase ABA2 expression and lead to larger plantsize or other unexpected phenotypes, ABA2 overex-pression transgenic plants were generated. The ABA2transgene in transgenic plants was driven by a 35Spromoter and contained the BAR gene for Basta her-bicide resistance as a selectable marker. More than 10independent transgenic lines were isolated on the ba-sis of herbicide resistance. Three homozygous transgenic

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lines (4-3, 4-4, and 5-1) were randomly chosen forfurther study. Under normal growth conditions in soil,the overexpression lines exhibited no apparent phe-notypic differences in aerial structures from the wildtype, but the aba2 mutant displayed a typical mutantphenotype, with small plant size and early flowering(Fig. 1A).

Southern-blot analysis further demonstrated thatthese three transgenic lines contained three or fourcopies of the T-DNA insertion (data not shown). Re-verse transcription (RT)-PCR revealed that these lineshave considerably higher levels of ABA2 transcriptsthan the wild type when grown in soil (Fig. 1B), inliquid culture, or on agar plates (data not shown). TheABA2 transcript shown in the aba2 mutant was slightlyshorter than that of wild type and transgenic linesbecause of the 53-bp deletion in the mutant (Fig. 1B).

Transgenic Plants Overexpressing ABA2 DelaySeed Germination

Because the level of ABA content controls seed dor-mancy, we tested whether ABA2 overexpression linesmight increase the ABA level and result in deeper seeddormancy. As shown in Figure 2, germination of thethree transgenic lines was delayed relative to that ofthe wild type without cold pretreatment. For instance,after 2 d, wild type had 93.7% 6 3.5% seed germina-tion, whereas transgenic lines 4-3, 4-4, and 5-1 hadonly 55.4% 6 8%, 51.8% 6 2.7%, and 36.5% 6 2.3%

germination, respectively (Fig. 2A). With cold pre-treatment for 3 d at 4�C, the germination delay fortransgenic lines was impaired after 2-d growth on agarplates with 1% Suc (Fig. 2B).

A similar seed germination delay in overexpressionlines was also shown on 2% and, to a lesser extent, on6% Glc agar plates (Fig. 2, C and D). As expected, theABA-deficient mutant aba2 always germinated earlierthan the wild type and the overexpression linesthroughout the germination period tested. The datasuggest that the delayed germination pattern shown inthe overexpression lines is reduced at the higher sugarconcentration. Compared to seeds grown on 1% Suc,those grown on 2% Glc had a lower germination rate.Low sugar concentration promotes, but high sugarconcentration inhibits, seed germination (Zhou et al.,1998; Arenas-Huertero et al., 2000; Finkelstein andLynch, 2000). Delayed germination was observed in allgenotypes on 6% Glc as compared to 2% Glc. For in-stance, 6-d germination rates on 6% Glc for aba2, wildtype, and transgenic overexpressing lines 4-3, 4-4, and5-1 were 88.9% 6 5.9%, 32% 6 2.8%, 19.8% 6 4.6%,18% 6 4.2%, and 24.3% 6 7.4%, respectively; however,germination rates over 90% were observed in allgenotypes on 2% Glc. It is noteworthy that wild typeand overexpression lines showed seed germinationfollowed by developmental arrest on 6% Glc (Fig. 2E).This observation can be explained by Glc-induced ABAaccumulation that further suppresses seedling growthand development. However, the ABA-deficient mu-tant aba2 lacks such inhibition and grows steadily anddevelops true leaves (Cheng et al., 2002). This notionwas further confirmed by the addition of fluridone, anABA biosynthesis inhibitor, to the medium; wild typeand ABA2 overexpression lines showed an aba2 phe-nocopy, with root elongation, cotyledon expansion,and true leaf development on fluridone-containing me-dium (Fig. 2E). Thus, the developmental arrest dis-played on 6% Glc is most likely due to de novo ABAbiosynthesis and accumulation. Fluridone is a selec-tive inhibitor of carotenoid synthesis, which interfereswith desaturation steps of carotenoid biogenesis andfurther blocks the accumulation of carotenoids. Thus,fluridone treatment inhibits carotenoid biosynthesisand leads to an albino-like seedling phenotype due tophotooxidation of chlorophyll (Henson, 1984).

Effect of ABA on Primary Root Growth

Although ABA2 transgenic plants showed no visibledifferences from wild type at aerial parts (Fig. 1A), ourprevious data showed strong ABA2 expression in roottissue (Cheng et al., 2002). This led us to examine theeffect of ABA on underground tissue in wild-type,aba2, and transgenic plants. As shown in Figure 3, with11-d growth on 1% Suc, the aba2 mutant had a shorterprimary root length; however, the primary root lengthin three transgenic lines had no significant differencefrom wild type (Fig. 3, A and B). Quantitative analysisdemonstrated that the average length of newly grown

Figure 1. Isolation and characterization of ABA2 overexpression lines.A, Phenotypic comparison of the wild type, aba2, and overexpressionline (4-3) grown in soil for 21 d. B, RT-PCR results of wild type, aba2, andoverexpression lines (4-3, 4-4, and 5-1). Total RNA was isolated fromseedlings grown in soil for 21 d. Two independent experiments wereperformed, each with a duplicate and with consistent results. WT, Wildtype; UBQ, ubiquitin. [See online article for color version of this figure.]

Expression of AtABA2/GIN1 in Response to Stress

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primary roots in wild type, aba2, and three transgeniclines (4-3, 4-4, and 5-1) was 6.0 6 0.17, 4.74 6 0.19,5.92 6 0.19, 6.02 6 0.28, and 5.93 6 0.19 cm, respec-tively (Fig. 3B). Further ABA immunoassays demon-strated that transgenic plants grown on 1% Suc for 11 dhad elevated levels of ABA, on average approximately44.7% higher than wild type, whereas the aba2 mutanthad the lowest level of ABA, approximately 27% of thewild-type level (Fig. 3C). The average elevated ABAlevel in these three transgenic lines is statistically sig-nificant, with P , 0.05 (P 5 0.023), Student’s t test. Ifthe reduction of primary root elongation shown in themutant is due to ABA deficiency, exogenous ABAapplication will restore the root length of the mutant tothat of wild type. As expected, addition of a smallamount of ABA (25–50 nM) promoted primary rootelongation in the mutant, with an average length close

to that of wild type and transgenic lines (Fig. 3B). At500 nM ABA, the root lengths of wild type, mutant, andtransgenic lines were similar, but slightly reduced ascompared with the length after application with theother two ABA concentrations. The seedlings displayedretarded growth with applications of more than 500 nM

ABA (data not shown). To test whether the effect ofABA on primary root elongation is ecotype dependent,we also assayed another aba2 allele, glucose insensitive1-1 (gin1-1; Wassilewskija background). gin1-1 alsohad a shorter primary root length than its wild type(Fig. 3D). The average length of newly grown primaryroots for the wild type and mutant was 7.68 6 0.40 and6.33 6 0.38 cm, respectively. A similar result was alsoobserved between gin1-2 and its corresponding wildtype, Landsberg erecta (data not shown). Again, exog-enous application of ABA (25–500 nM) restored the

Figure 2. ABA2 overexpression lines delay seed germination. A, Germination of wild type and overexpression lines without coldtreatment. Seeds were grown on 1% Suc agar plates for the times shown. Radicle emergence over 1 mm is referred to asgermination. B, Germination of wild type and overexpression lines with cold pretreatment for 3 d at 4�C. The seed growth conditionwas the same as in A. C, Germination of wild type, aba2, and overexpression lines on 2% Glc agar plates. D, Germination of wildtype, aba2, and overexpression lines on 6% Glc agar plates. E, Phenotypes of wild type, aba2, and overexpression lines grown on6% Glc agar plates with or without 1 mM fluridone for 14 d. Germination rates from A to D were counted as germinated seeds/totalgerminated seeds at day 10. The results shown were the means 6 SD of three independent experiments using different seed batches,with consistent results, each with 200 to 250 seeds. The seeds in B, C, D, and E were subjected to cold pretreatment at 4�C for 3 dbefore planting; in A, without cold treatment. WT, Wild type. [See online article for color version of this figure.]

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gin1-1 primary root length to near that of its wild type.Taken together, the results indicate that the reductionof primary root length shown in the mutant is primar-ily due to ABA deficiency, regardless of ecotype. Inaddition, ABA2 overexpression lines with elevatedABA levels did not show a longer primary root length,which reflects that basal levels of ABA are essential formaintaining primary root elongation.

Effect of Osmoticum on Seedling Growthand Development

It has been reported that overexpression of NCED3causes elevated levels of ABA and enhances plant tol-erance to drought in Arabidopsis (Iuchi et al., 2001)and tobacco (Qin and Zeevaart, 2002). To test whetherABA2/GIN1 overexpression lines with increased levelsof ABA also show tolerance to drought, we grew seed-lings on a polyethylene glycol (PEG)-infused mediumthat created osmotic stress mimicking drought condi-

tions. Several PEG concentrations were tested (12.5%,25.0%, 40.0%, 55.0%, and 70.0%). Seedlings grown on40% PEG-infused medium (20.7 MPa Cw, according toVerslues and Bray, 2004) consistently displayed differ-ential growth rates among wild type, aba2, and theABA2-overexpressing lines. Seedling growth wasstrongly inhibited on medium with greater than 40%PEG (data not shown). Seedling growth in one set ofexperiments is shown in Figure 4A. Wild type had ahigher bleached cotyledon ratio (41.1% 6 6.9%) thanoverexpression line 4-4 (27.4% 6 3.9%; Fig. 4B),suggesting that transgenic plants may be more tolerantto low water potential than wild type.

It is surprising that the aba2 mutant had the lowestdamage (11.7% 6 3.8%) under the same growth con-ditions. Similar results were also observed in anotherABA-deficient mutant, sto1/nced3, versus its corre-sponding wild type (C24; data not shown). The reasonfor this discrepancy remains to be illustrated. One ofthe possibilities might be related to an increase in

Figure 3. Effect of ABA on primary root elongation. A, Wild type, aba2, and overexpression lines 4-3, 4-4, and 5-1 were grownon 1% Suc agar plates for 5 d, then transferred to the same medium with or without ABA treatment and grown vertically foranother 6 d. Arrowheads indicate the position of primary root tips. B, Primary root length of wild type, aba2, and overexpressionline seedlings grown on 1% Suc for 5 d, then transferred to the same medium with or without ABA and grown vertically foranother 6 d. The values are the means 6 SD of 10 to 12 seedlings of each genotype. The experiment was repeated twice withconsistent results. C, ABA content of wild type, aba2, and overexpression lines. Seedlings were grown on 1% Suc agar plates for11 d and then subjected to ABA immunoassay. The values are the means 6 SD of three independent experiments using differentseed batches, each performed in duplicate. WT, Wild type. D, Primary root length of wild type (Ws) and gin1-1. Seedlings had thesame growth conditions as in B, except with vertical growth for 7 d. The values are the means 6 SD of 12 seedlings of eachgenotype. The experiment was repeated twice with consistent results. The seeds in A to D were subjected to cold pretreatment at4�C for 3 d before planting on agar plates. [See online article for color version of this figure.]





stomatal aperture under such conditions. Ruggieroet al. (2004) reported that the sto1/nced3 mutant’s sto-matal aperture at day and night is larger than that ofwild type under high humidity conditions. This find-ing might explain why the aba2 mutant had a highersurvival rate because of its efficient absorption ofwater vapor from the air within the high-humiditypetri dish. We also observed that wild-type and trans-genic plants showed anthocyanin accumulation innewly growing true leaves with a slower growth rate,whereas the aba2 mutant retained light greening with ahigher growth rate or had a larger plant size on aver-age under low-water-potential conditions (Fig. 4A).

Effect of Salinity on Germination and Growth of ABA2Overexpression Lines

ABA has long been believed to be a stress-relatedphytohormone whose biosynthesis or level can be in-

duced by various stresses. To further determine whetherthe seed germination delay shown on sugar mediumalso occurs under salinity stress, we grew seeds on 1%Suc agar plates with or without NaCl. As shown withsugar treatments, the aba2 mutant showed early ger-mination regardless of the presence or absence of NaCl(compare Figs. 2 and 5). For instance, the aba2 mutantbegan germination within 24 h with 125 mM NaCl and48 h with 250 mM NaCl, whereas germination of wildtype and transgenic lines was delayed until 24 h with125 mM NaCl (Fig. 5A) and 72 h with 250 mM NaCl(Fig. 5B). The higher NaCl concentration delayedseed germination in all genotypes. Transgenic plantsshowed a slight germination delay relative to wildtype at 125 mM NaCl (Fig. 5A), whereas transgeniclines germinated in a manner similar to the wild typeat 250 mM NaCl (Fig. 5B). At 125 mM NaCl, germina-tion rates within the first 2 d for aba2, wild type, 4-3,4-4, and 5-1 were 90.8% 6 1.8%, 76.8% 6 3.9%,65.6% 6 8.1%, 57.7% 6 6.8%, and 54.2% 6 2.4%, re-spectively. At 250 mM NaCl, the aba2 mutant displayedexpanded cotyledons with light greening, but no trueleaf development after 7 d. In contrast, wild type andoverexpression lines germinated and then showeddevelopmental arrest (Fig. 5C). The light greeningcotyledons in aba2 became bleached after 18 d of ger-mination; the bleached seedlings were not viable afterbeing transferred to fresh 1% Suc medium withoutNaCl, whereas the arrested seedlings of wild type andoverexpression lines restored their green cotyledonsand normal true leaf growth thereafter (data notshown). This observation indicates that wild typeand overexpression lines have a self-protecting abilitymodulated by ABA and inhibited further growthand development under high NaCl conditions. NaCl-induced developmental arrest can be overcome with1 mM fluridone: Wild type and overexpression linesshowed the aba2 phenocopy with expanded cotyle-dons (Fig. 5C). The result indicates that NaCl-induceddevelopmental arrest is most likely due to de novoABA biosynthesis and accumulation.

Furthermore, immunoassays were used to deter-mine ABA levels in seedlings from each genotypetreated with 125 and 250 mM NaCl, respectively. Theresults exhibited that the aba2 mutant had only 45.3%(Fig. 5D) and 45.7% (Fig. 5E) of the wild-type ABAlevels at 125 and 250 mM NaCl, respectively. How-ever, transgenic lines were, on average, approximately1.74- and 2.11-fold (Fig. 5, D and E, respectively)higher in ABA levels than that of wild type at 125and 250 mM NaCl, respectively. The average increaseof ABA level in these three transgenic lines is statis-tically significant, with P , 0.05 (P 5 0.024 at 125 mM

NaCl and P 5 0.025 at 250 mM NaCl), Student’s t test.Surprisingly, transgenic seedlings with high ABAlevels did not show a germination delay when grownon 250 mM NaCl. Thus, the data suggest that there aredistinct mechanisms that may impair or overcomeABA-mediated inhibition of seed germination undersevere salinity stress.

Figure 4. Effect of osmoticum on seedling growth. A, Seeds with coldpretreatment were grown on 1% Suc agar plates for 5 d, then transferredto the same fresh medium with 40% PEG-infused treatment for another4 weeks. WT, Wild type. B, Quantification of bleached cotyledonsderived from A. The values are the means 6 SD of three independentexperiments, each genotype with 100 seedlings per experiment. [Seeonline article for color version of this figure.]

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Phenotypic comparison revealed that the aba2 seed-lings at 200 mM NaCl had lighter greening and glassierleaves than wild type and overexpression line seed-lings (Fig. 5F). When grown on 1% Suc agar platescontaining various NaCl concentrations (125–200 mM),seeds may germinate and develop true leaves; uponprolonged culture, however, the seedlings becamebleached and died. As shown in Figure 5, F and G,

although the aba2 mutant revealed early germinationon NaCl-containing medium, more aba2 bleached seed-lings were observed after prolonged culture. For in-stance, with 200 mM NaCl, wild type, aba2, and ABA2overexpression lines had bleached seedling frequen-cies of 55.8% 6 3%, 81.6% 6 5.7%, and 39.3% 6 9.6%,respectively. In soil, after 21-d-old seedlings werewatered with increasing concentrations of NaCl (50,

Figure 5. Effect of salinity on germination and plant growth. A and B, Germination of wild type, aba2, and overexpression lines on1% Suc agar plates supplemented with 125 (A) or 250 mM (B) NaCl. Germination rates were the means 6 SD of three independentexperiments using different seed batches, with consistent results, each with 150 to 200 seeds. WT, Wild type. C, Phenotypiccomparison of wild type, aba2, and overexpression lines grown on 1% Suc agar plates plus 250 mM NaCl with or without 1 mM

fluridone for 14 d. D and E, ABA levels of wild type, aba2, and transgenic lines. Seedlings were grown on 1% Suc agar plates for 5 d,then transferred to the same fresh medium supplemented with 125 mM NaCl for another 7 d (D) or 250 mM NaCl for another 2 d (E).The values are the means 6 SD of three independent experiments using different seed batches, each performed in duplicate. F,Phenotypic comparison of wild type, aba2, and transgenic line 4-4. Seedlings were grown on 1% Suc plates supplemented with200 mM NaCl for 28 d. G, Quantification of bleached plants of wild type, aba2, and transgenic line 4-4 in F. The values are themeans 6 SD of three independent experiments using different seed batches and with consistent results, each with 200 to 250 seeds.H, Phenotypic comparison of wild type, aba2, and overexpression line 4-4 in response to salinity. A total of 75 plants from eachgenotype, grown in soil for 21 d, were tested by watering with four increasing concentrations of NaCl (50, 100, 150, and 200 mM),each for 4 d for a total of 16 d. Seeds tested in this study were cold pretreated before planting on agar plates or in soil.





100, 150, and 200 mM NaCl), each for 4 d for a total of16 d, the aba2 mutant became bleached (Fig. 5H). How-ever, the wild type had just begun to bleach and thetransgenic line had only dark greening rosette leaveswith anthocyanin accumulation. These results indicatethat ABA2 overexpression lines were more tolerantto salinity than wild type when they were grown onagar plates or in soil. Moreover, tolerance to salinityis associated with elevated ABA levels in transgeniclines.

ABA2 Promoter Activity in Response to Stresses

Our previous data showed that ABA2 is a uniquegene because its regulation is distinct from other ABAbiosynthetic genes in response to stresses. For in-stance, ABA1, NCED3, and AAO3 transcripts are rap-idly induced by dehydration and ABA treatment for3 h, but ABA2 displays no conceivable change (Chenget al., 2002). To test whether ABA2 expression is up-regulated by stresses, we used a more sensitive test(i.e. b-glucuronidase [GUS] activity) to determine ABA2promoter activity. Two transgenic lines harboring theABA2 promoter and GUS reporter gene (ABA2TGUS),each in wild-type and aba2 backgrounds, were usedto assay ABA2 promoter activity in response to stress.Southern-blot analysis indicated that transgenic line4-12 (wild-type background) had at least two transgeneinsertions and the remaining lines had at least threetransgene copies (data not shown). The ABA2 promoterregion was approximately 2.9 kb upstream of the ATGstart codon and had been used to complement the aba2mutant to restore the wild-type phenotype (Chenget al., 2002). Thus, this promoter region contains all orat least most of the cis-acting elements that are essentialfor controlling ABA2 gene expression.

To determine ABA2 promoter activity under rapiddehydration, aerial parts of tissues from transgenicplants grown in soil for 21 d were removed and placedin an electronic dry box with 40% relative humidity for1.5 and 3 h. As shown in Figure 6A, 1.5-h dehydrationonly slightly reduced ABA2 promoter activity as com-pared to control 1.5 h. These reductions were relativelysmall, about 28%, 38%, 19%, and 43% of the controlactivity for lines 1-11 (wild-type background), 4-12(wild-type background), 3-6 (aba2 background), and3-12 (aba2 background), respectively. However, 3-hdehydration showed no significant change of ABA2promoter activity in each line as compared to that ofthe 3-h control. ABA2 promoter activity was consider-ably higher after 4 d of withholding water than that ofthe control; for instance, the transgenic line 1-11 (wild-type background) under 4-d drought had 16-foldhigher activity than that of the control; strikingly,activity in the transgenic plant line 3-12 (aba2 back-ground) under 4-d drought was 38.6-fold higher thanthat of the control. Three-day cold treatment alsoinduced promoter activity higher than that of thecontrol, but in transgenic lines 1-11 (wild-type back-ground) and 3-6 (aba2 background) the activity was

Figure 6. ABA2 promoter activity in response to diverse stress. A,Determination of GUS activity under drought condition. Aerial parts oftissues were removed from 21-d-old plants grown in soil and imme-diately placed on an electronic dry box with 40% relative humidity for1.5 and 3 h. B, Determination of GUS activity under diverse stress.Plants were grown in soil for 21 d and subsequently treated with cold(4�C; 3 d), drought (4 d), flooding (1 d), and salt stresses (125 and250 mM NaCl, each for 1 d), respectively. Control samples were harvestedat 21 dap, at the beginning of stress treatments. C, Determination ofGUS activity under Suc and NaCl treatments. Seedlings were grown on1% Suc agar plates with or without 125 or 250 mM NaCl for 14 d.Results in A, B, and C are the means 6 SD of three independent ex-periments, each experiment with a duplicate. Transgenic plants har-boring the ABA2TGUS transgene were under wild-type (lines 1-11 and4-12) and aba2 mutant (lines 3-6 and 3-12) backgrounds.

Lin et al.




reduced by approximately 60% and 26%, respectively,as compared to that of 4-d drought. Although thecontrol samples shown here (Fig. 6B) were harvestedat 21 days after planting (dap), at the beginning ofstress treatments, we also harvested the control sam-ples at the same time points as the stress-treatedsamples. The results turned out to have very smallvariation 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 activitychange all below 0.49-, 0.43-, and 1.0-fold for everytransgenic line tested relative to that of the controlsamples at day 0 (21 dap; data not shown). Becausethese variations were very small compared to the var-iations between stress-treated and control samples atday 0 (Fig. 6B), we believe that there was no significantdevelopmental effect on ABA2 promoter activityamong these control samples during this 4-d develop-ment (i.e. 21–25 dap), at least in this case.

For NaCl treatments, transgenic plants grown in soilfor 3 weeks were soaked with water, 125 or 250 mM

NaCl for 24 h; water was sufficient to induce ABA2promoter activity in transgenic plants (both wild-typeand aba2 background), albeit with less efficiency thanwith NaCl and drought treatment (Fig. 6B). As waterflooding may cause a submergence or hypoxia effect,we observed that the ABA2 promoter was submer-gence inducible (data not shown). Transgenic plants(wild-type background) with NaCl treatment had arelative induction of ABA2 promoter activity with avalue higher than with cold (4�C) treatment, butslightly lower than with 4-d drought treatment. After14 d on agar plates, ABA2 promoter activity in both thewild-type and aba2 backgrounds was induced by NaCltreatment. Such induction was more pronounced withthe addition of 125 mM NaCl in transgenic plants withaba2 background (Fig. 6C). As compared with 1% Suctreatment, the addition of NaCl increased promoteractivity 3.2-fold at 125 mM and 6.9-fold at 250 mM NaClin line 1-11 (wild-type background); activity in line3-12 (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 (aba2background) are most likely due to low metabolism at250 mM NaCl treatment for 14 d. At that time point,some transgenic plants in the mutant backgroundstarted showing a bleached phenotype, whereas trans-genic lines in the wild-type background remainedwith developmental arrest. Taken together, these find-ings indicate that ABA2 promoter activity was up-regulated by NaCl stress.

In general, ABA2 promoter activity is remarkablyinduced by prolonged cold, drought, flooding, andsalinity, but not short-term dehydration. Induction isprofound in transgenic lines with an aba2 background.It indicates that the feedback inhibition of ABA2 pro-moter activity by ABA is likely missing in the ABA-deficient mutant aba2. Thus, mutants may build upconsiderable levels of ABA2 promoter activity relativeto transgenic lines under a wild-type background.

Up-Regulation of ABA- and Sugar-Related Genes

in Response to Drought

As mentioned above, because ABA2 promoter ac-tivity was considerably changed under prolongedstress conditions but not short rapid stress treatment,ABA2 expression is controlled differently from otherABA biosynthetic genes that show early stress re-sponse. One of the possibilities is likely due to theprimary metabolic change during prolonged stresstreatments, which in turn regulate ABA2 expression.Because sugars play a central role in plant growthand development and ABA biosynthetic genes areup-regulated by sugars, we tested for alterations insugar-related gene expression under prolonged stressconditions at the molecular level. As shown in Figure7, the ABA2 transcript was overexpressed in trans-genic plants grown in soil for 3 weeks followed bydrought treatment for 4 d or without drought treat-ment. The levels of the ABA2 transcript were elevatedin the wild type after 4-d drought treatment, in agree-ment with the induction of ABA2 promoter activity(Fig. 6B). However, because the aba2 transcript has a53-bp deletion, its stability might be labile so thatthe accumulation of the aba2 transcript was not as highas its promoter activity in response to 4-d drought.Similarly, the NCED3 (At3g14440) transcript was alsoinduced under the same growth conditions. It is inter-esting that the aba2 mutant accumulated a higheramount of NCED3 transcript than that of wild-typeand transgenic plants. The reason for this accumula-tion remains unknown. One of the possibilities is thatNCED3 expression is inducible by ABA and droughtmay increase ABA biosynthesis and accumulation;however, the aba2 mutant lacks ABA under droughtconditions and might also result in a loss of negativefeedback regulation of NCED3 expression.

For sugar-related genes, AtHXK1 (At4g29130), asugar sensor, and AtSusy1 (At5g20830), a Suc-cleavingenzyme, were both up-regulated under stress condi-tions, whereas AtSusy2 (At5g49190) showed low toundetectable levels (data not shown). The up-regulationpatterns of AtHXK1 and AtSusy1 expression in ABA2transgenic plants under drought conditions were im-paired or unchanged as compared to the wild type andaba2 mutant. Thus, these results provide evidence thatlate expression of ABA2 in response to drought, at leastin part, is associated with up-regulated expression ofsugar-related genes.

DISCUSSION

Overexpression of ABA2 in Arabidopsis Increases ABAContent and Promotes Seed Germination Delay and

NaCl Tolerance

It has been reported that overexpression of ABAbiosynthetic genes results in an elevated ABA level andextended seed dormancy under nonstress conditions(Frey et al., 1999; Thompson et al., 2000b; Iuchi et al.,





2001; Qin and Zeevaart, 2002). Similar results were alsoseen in this study when ABA2 overexpression trans-genic seeds without precold treatment were grown onagar plates with 1% Suc. ABA2 overexpression trans-genic seeds showed germination delay as compared towild type. We observed that exogenous application ofthe ABA biosynthesis inhibitor fluridone promotedseed germination but did not change germinationpatterns. It suggests that the germination pattern isaffected by the preexistence of ABA in germinatingseeds. In addition, ABA2 expression was detectable at24 h of seed germination via GUS staining of ABA2TGUS (line 1-11) transgenic seeds (data not shown).ABA2 transgenic plants also had ABA accumulation atdifferent developmental stages (see below). Thus,transgenic seeds with germination delay were mostlikely due to ABA2 overexpression, resulting in theincrease of ABA content in transgenic seeds. In addi-tion, transgenic seedlings also had an approximately44.7% increase of ABA levels as compared to wild typewhen grown on 1% Suc agar plates. The increase ofABA levels in transgenic lines was much more pro-nounced under high NaCl conditions (Fig. 5D),whereas the germination delay was impaired or abol-ished when transgenic seeds were grown on 125 (Fig.5A) or 250 mM NaCl (Fig. 5B). Thus, these data suggestthat there are distinct mechanisms by which ABA-mediated inhibition of seed germination is attenuatedor overcome under severe stress. The mechanismscausing an antagonistic effect on ABA inhibition ofseed germination remain to be illustrated. One of thepossibilities is the induction of ethylene productionthat is also stress inducible and has an antagonisticeffect on ABA action (Zhou et al., 1998). Alternatively,we still cannot exclude the possible effect of osmoticand ion toxicity, which might reduce or override ABAinhibition of seed germination at high NaCl conditions.

Expression of ABA2 in Response to Stress

It is believed that plants have multiple stress per-ceptions and signal transduction pathways to generatespecific and common responses to stresses. Althoughthe common response may have cross talk at varioussteps in the pathways, the specific response mayreflect the unique pathway induced only by specificstresses (for review, see Chinnusamy et al., 2004).Thus, germinating seeds may generate a unique signalpathway in response to high Glc and NaCl stresses,which results in different seed germination patternsand seedling phenotypes. We also observed that theaddition of the ABA biosynthesis inhibitor fluridoneinto the medium enhanced seed germination, but didnot change the germination patterns under 6% Glc and250 mM NaCl (data not shown), which indicates thatthe different germination patterns seen with Glc andNaCl stresses are most likely determined prior to denovo ABA biosynthesis and/or accumulation duringseed germination. It is interesting that, despite theirdifferent effect on seed germination, both Glc andNaCl stresses may induce postgermination develop-mental arrest in wild type and overexpression lines,but not the aba2 mutant that displayed near-normalgrowth and expanded cotyledons with no true leafdevelopment on 6% Glc and 250 mM NaCl, respec-tively. Developmental arrest can be overcome by theapplication of the ABA biosynthesis inhibitor fluri-done, the treated seedlings displaying the aba2 mutantphenocopy (Figs. 2E and 5C). Thus, postgerminationdevelopmental arrest is predominantly due to de novoABA biosynthesis and accumulation. In addition toelevated ABA levels and extended seed dormancy,transgenic plants overexpressing ABA2 also showedmore tolerance to NaCl stress than wild type whengrown on agar plates and/or in soil. Thus, this studyprovides extensive evidence that manipulation ofABA2 expression in Arabidopsis increases the ABAlevel, deepens seed dormancy, and increases NaCltolerance. This finding is useful and can be extended toother important agricultural crops for improvingstress tolerance.

ABA with a Function in Maintaining PrimaryRoot Elongation

In addition to its function as a stress hormone, ABAhas long been considered a plant growth inhibitorbecause the elevated endogenous ABA content in-duced by stress or exogenous ABA application nor-mally suppresses vegetative tissue growth. However,most ABA-deficient mutants have a smaller size thanthe wild type, which reflects that the basal level ofABA is essential for normal plant growth and devel-opment. It is interesting that all overexpression lines ofABA biosynthetic genes reported to date, includingthose in this study, show a phenotype not differentfrom that of wild type in aerial parts under normalgrowth conditions. However, because ABA2 is a unique

Figure 7. Expression of ABA- and sugar-related genes in response todrought. Plants were grown in soil for 3 weeks, and then subjected towithholding water for 4 d (drought). Control samples were harvested atthe same time as drought-treated samples. Three independent exper-iments were performed, except for control with two independentexperiments, each with a duplicate and with consistent results. WT,Wild type; HXK, hexose kinase; Susy, Suc synthase; UBQ, ubiquitin.

Lin et al.




gene, its regulation and response to stresses are dif-ferent from other ABA biosynthetic genes in someaspects. In addition, ABA2 tissue-specific expression ispredominantly restricted to vascular bundles in manytissues, including roots. The latter compelled us toexamine the effect of ABA on root growth.

Our data demonstrate that the aba2 mutant had ashorter primary root length than that of wild-type andtransgenic plants (Fig. 3, A and B). The reduction isprimarily due to ABA deficiency because exogenousapplication of ABA into the medium restored the rootgrowth in the mutant to normal. We observed that ABAsensitivity differs in distinct ecotypes, to some extent,but the short primary root length caused by the lack ofABA was ecotype independent (Fig. 3, B and D). It isnoteworthy that primary root elongation was not inhib-ited by low amounts of exogenous ABA (less than 500nM); with more than 500 nM ABA, plants normally showretarded growth and have inhibited primary root elon-gation and decreased lateral root numbers. ABA in-hibits radicle emergence, but not seedling growth;however, such an inhibitory effect can be suppressedwith the presence of low concentrations of sugar(Finkelstein and Lynch, 2000). De Smet et al. (2003)also reported that ABA induces lateral root develop-mental arrest at a specific stage and subsequentlyblocks lateral root meristem formation. Consistentwith this notion, all exogenous ABA concentrationstested in this study, except for 25 to 50 nM, inhibitedlateral root growth and development (data not shown).A genetic approach also reveals that ABA promotesprimary root elongation in maize under low-water-potential conditions (Sharp et al., 1994). Here, weextensively studied ABA2 overexpression lines. Theresults showed that the primary root length in ABA2overexpression lines was similar to wild type with orwithout ABA treatments. These data reflected thefunction of basal levels of ABA in maintaining normalprimary root growth. Thus, our current data furtherconfirm the dual function of ABA in plant growth anddevelopment (Cheng et al., 2002); that is, the basal levelor biophysiological concentrations of ABA may func-tion as a plant growth promoter under normal growthconditions and as a plant growth inhibitor understressful conditions, which may also be an adaptiveresponse beneficial to the plant during recovery.

Up-Regulation of ABA2 Expression through Long-Term,But Not Short-Term, Stress Treatments

Our previous data demonstrated that ABA2 is aunique gene whose regulation is different from otherABA biosynthetic genes, such as AtABA1, AtNCED3,AtABA3, and AAO3, in response to stresses. The lattergenes are transcriptionally up-regulated under short-term drought conditions (#3 h), whereas ABA2 is not.The current study further confirms that ABA2 pro-moter activity had little change after a short period ofdrought treatment (#3 h; Fig. 6A), which is consistentwith no conceivable change in the level of ABA2 tran-

scripts under the same conditions (Cheng et al., 2002;Gonzalez-Guzman et al., 2002). However, prolongedstressful treatments led to a relatively stronger induc-tion of ABA2 promoter activity (Fig. 6, B and C) anddetectable induction of the ABA2 transcript (Fig. 7).

The reason for the discrepant regulation betweenABA2 and other ABA biosynthetic genes is unknown.Analysis of the ABA2 promoter within 2.8 kb upstreamof the translational start site (ATG) using the Arabidop-sis Gene Regulatory Information Server (agris; OhioState University; http://Arabidopsis.med.ohio-state.edu/AtcisDB) and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare; Lescot et al., 2002)revealed that the ABA2 (At1g52340, chromosome1 location: 19493468–19495317) promoter possessesthree ABA response element (ABRE)-like elements(TACGTGTC, TACGTG, and GCCACGTGT at 636,708, and 1,418 bp upstream of ATG, respectively). Incontrast, the AtNCED3 (At3g14440, chromosome 3 lo-cation: 4831295–4833606) promoter contains one dehy-dration response element (DRE; TACCGACAT, at 1,632bp upstream of ATG), two ABRE (CACGTGGC at 207and 2,372 bp upstream of ATG), and three ABRE-likeelements (TACGTG, TACGTG, and GACCGTA at 807,1,945, and 2,163 bp upstream of ATG, respectively).Thus, AtNCED3 with a DRE and more copies of ABREand ABRE-like elements than that of ABA2 may re-spond to drought and other stresses more rapidly andefficiently. Another possibility is that the prolongedstressful conditions might change the primary metab-olic features, such as elevated soluble sugars (Dejardinet al., 1999), which in turn regulate ABA2 expression.Evidence that supported this hypothesis was the ele-vated expression of sugar-related genes such asAtHXK1 and AtSusy1 under prolonged stress (Fig. 7;Dejardin et al., 1999). These two enzymes may generatethe soluble sugars hexose-P, and UDP-Glc and Fru,respectively, to adapt plants to the stress environment.Our previous data also demonstrate that increasedexogenous sugar concentration stimulates ABA2 geneexpression and causes an elevated ABA level. Theinduction, however, is impaired or abolished in the aba2mutant (Cheng et al., 2002). Thus, ABA2 is most likely alate expression gene in response to stresses, as com-pared with other ABA biosynthetic genes, and it mighthave a fine-tuning function in maintaining ABA levels inresponse to stresses through regulation of primarymetabolic changes. A high Glc concentration also in-duces expression of ABI genes, such as ABI3, ABI4, andABI5 (Cheng et al., 2002). Thus, sugar regulation ofplant growth and development may, at least in part, actthrough modulation of ABA biosynthesis and respon-sive gene expression.

MATERIALS AND METHODS

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). This mutant is also allelic to

san3, sis4, isi4, and sre1 isolated by other groups (Laby et al., 2000; Quesada

et al., 2000; Rook et al., 2001; Gonzalez-Guzman 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 mE s21 m22. 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) medium composed of one-half-strength Murashige and Skoog basal

salts, B5 organic compounds (Gamborg et al., 1968), and 0.05% MES. For

experimental treatments, the medium was supplemented with Glc, Suc, NaCl,

herbicide (glufosinate ammonium; Riedel-deHaen, catalog no. 45520), ABA

(Sigma; catalog no. SI–A1049), or fluridone (Wako; catalog no. 068–03081) at

concentrations listed in the ‘‘Results’’ section.

Transgene Constructs and Transgenic Plant Isolation

For overexpression, ABA2 (GIN1) full-length cDNA was amplified by

RT-PCR and cloned into pGEM-T Easy vector (Promega), followed by sub-

cloning into the binary vector (pSMAB704) driven by a constitutive 35S pro-

moter (35STABA2). 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 ABA2TGUS

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). T1 seeds with

cold pretreatment were screened on 1% Suc agar plates containing 25 mg/L

herbicide, glufosinate ammonium. At least 10 homozygous lines resistant to

herbicide were obtained at the T3 or T4 generation. Three homozygous lines

were randomly chosen for further study.

Germination and Root Elongation Tests

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 verti-

cally for another 6 or 7 d.

Preparation of Low-Water-Potential Medium Plates

One-half-strength modified Murashige and Skoog medium (pH 5.7) with

0.7% Phyto agar (Duchefa Biochemie B.V.) was autoclaved. The sterilized me-

dium was cooled to 50�C to 60�C and then aliquoted into petri dishes (100- 3

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

($12 h). The excess PEG solution was then removed. The procedure essen-

tially followed the protocol of Verslues and Bray (2004).

NaCl and Dehydration Treatments

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). For dehydration, seeds with cold pretreatment

were grown in soil for 21 d and then plants were excised. The aerial parts of

tissues were placed on plastic weighing boats and kept in an electronic dry

box (model-DX-76; Taiwan Dry Tech Corp.) with approximately 40% relative

humidity. The fresh weights of tissues were measured at 0-, 1.5-, and 3-h time

points.

GUS Activity Assay

Cold-pretreated transgenic seeds harboring the ABA2TGUS 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 b-mercaptoethanol, 10 mM Na2EDTA, 0.1% sodium

lauryl sarcosine, 0.1% Triton X-100). After centrifugation at 13K rpm for 10 min

at 4�C, the supernatants were removed for further GUS assay, essentially

according to the protocol of Jefferson et al. (1987), using a DyNA Quant 200

fluorometer (Amersham-Pharmacia Biotech).

RT-PCR

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 mg 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 accord-

ing to the manufacturer’s protocol. Synthesized cDNA was used as a template

for PCR.

ABA Assay

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 immuno-

assay, 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). For ABA determination, ABA was quantified by ELISA (Phyto-

detek ABA kit; Agdia) according to the manufacturer’s protocol.

ACKNOWLEDGMENTS

We thank Dr. Charles M. Papa (Tajen University, PingTung, Taiwan, Repub-

lic 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 Agro-

biological Sciences, Tsukuba, Japan) for providing the binary vector

pSMAB704. We are also grateful to Dr. C.H. Kao and Ms. Y.T. Hsu (Depart-

ment 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.

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ectopic expression of abscisic acid 2/glucose … · et al., 2002) and inhibit ﬂowering (razem et...

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