Overexpression of a Protein Phosphatase 2C fromBeech Seeds in Arabidopsis Shows Phenotypes Related toAbscisic Acid Responses and Gibberellin Biosynthesis1

David Reyes, Dolores Rodrıguez, Mary Paz Gonzalez-Garcıa, Oscar Lorenzo, Gregorio Nicolas,Jose Luis Garcıa-Martınez, and Carlos Nicolas*

Departamento de Fisiologıa Vegetal, Centro Hispano-Luso de Investigaciones Agrarias, Facultad de Biologıa,Universidad de Salamanca, 37007 Salamanca, Spain (D. Reyes, D. Rodrıguez, M.P.G.-G., O.L., G.N., C.N.);and Instituto de Biologıa Molecular y Celular de Plantas, Universidad Politecnica de Valencia-ConsejoSuperior de Investigaciones Cientificas, 46022 Valencia, Spain (J.L.G.-M.)

A functional abscisic acid (ABA)-induced protein phosphatase type 2C (PP2C) was previously isolated from beech (Fagussylvatica) seeds (FsPP2C2). Because transgenic work is not possible in beech, in this study we overexpressed this gene inArabidopsis (Arabidopsis thaliana) to provide genetic evidence on FsPP2C2 function in seed dormancy and other plantresponses. In contrast with other PP2Cs described so far, constitutive expression of FsPP2C2 in Arabidopsis, under thecauliflower mosaic virus 35S promoter, produced enhanced sensitivity to ABA and abiotic stress in seeds and vegetativetissues, dwarf phenotype, and delayed flowering, and all these effects were reversed by gibberellic acid application. The levelsof active gibberellins (GAs) were reduced in 35S:FsPP2C2 plants, although transcript levels of AtGA20ox1 and AtGA3ox1increased, probably as a result of negative feedback regulation, whereas the expression of GASA1 was induced by GAs.Additionally, FsPP2C2-overexpressing plants showed a strong induction of the Responsive to ABA 18 (RAB18) gene.Interestingly, FsPP2C2 contains two nuclear targeting sequences, and transient expression assays revealed that ABA directedthis protein to the nucleus. Whereas other plant PP2Cs have been shown to act as negative regulators, our results support thehypothesis that FsPP2C2 is a positive regulator of ABA. Moreover, our results indicate the existence of potential cross-talkbetween ABA signaling and GA biosynthesis.

Abscisic acid (ABA) and GAs regulate several as-pects of plant growth showing antagonistic effects indifferent developmental processes, including seeddormancy or germination events (for review, seeKermode, 2005; Kucera et al., 2005).

Several components implicated in ABA signaling,mainly those regulating seed dormancy, have beenidentified in ABA-response mutants by isolating theaffected genes and their corresponding proteins in-volving a complex network of positive and negativeregulators, including kinases, phosphatases, and tran-scriptional regulators (for review, see Finkelstein et al.,2002; Abe et al., 2003). Protein phosphorylation/de-phosphorylation events have been investigated indetail in ABA-mediated processes involving severalwell-known protein kinases and phosphatases (Leungand Giraudat, 1998; Finkelstein et al., 2002), such as

PKABA1, an ABA-induced protein kinase that sup-presses GA-inducible gene expression in barley (Hor-deum vulgare) aleurone layers (Gomez-Cadenas et al.,1999, 2001); the guard cell-specific protein kinaseAAPK, necessary for stomatal closure mediated byABA (Li et al., 2000); and the protein kinase OST-1, anessential component mediating stomatal regulation inresponse to drought (Mustilli et al., 2002). Addition-ally, fourArabidopsis (Arabidopsis thaliana) Ser-Thr pro-tein phosphatase 2Cs (PP2Cs; ABI1, ABI2, AtPP2CA,and HAB1) and one PP2C from beech (Fagus sylvatica;FsPP2C1) have been described as negative regulatorsof the ABA signal transduction cascade (Gosti et al.,1999; Merlot et al., 2001; Tahtiharju and Palva, 2001;Gonzalez-Garcıa et al., 2003; Saez et al., 2004; Kuhnet al., 2006; Yoshida et al., 2006).

In addition, analysis of mutants with reduced pro-duction of GAs has demonstrated the role of thesehormones in several aspects of plant development,including stem elongation, fruit set, flower induction,or seed germination (for review, see Lange, 1998). Forinstance, Arabidopsis mutants blocked in GA biosyn-thesis fail to complete germination, this effect beingreverted by exogenous addition of GAs (Koornneefand van der Veen, 1980). Three different phases can beconsidered within the biosynthetic pathway of GAs(Olszewski et al., 2002). The first one is the formationof ent-kaurene, involving geranylgeranyl diphosphatecyclation via copalyl diphosphate as an intermediate.

1 This work was supported by the Ministerio de Ciencia yTecnologıa, Spain (grant nos. BFI2003–01755 and BIO2003–00151),by Junta de Castilla y Leon (grant no. SA046A05), and by a ‘‘Ramon yCajal’’ research contract (to O.L.).

* Corresponding author; e-mail cnicolas@usal.es; fax 34–923294682.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:Carlos Nicolas (cnicolas@usal.es).

Article, publication date, and citation information can be found atwww.plantphysiol.org/cgi/doi/10.1104/pp.106.084681.

1414 Plant Physiology, August 2006, Vol. 141, pp. 1414–1424, www.plantphysiol.org � 2006 American Society of Plant Biologists

Ent-kaurene is then converted to GA12 and GA53 (a C-13hydroxylated GA) by oxidative reactions catalyzed byP450-dependent monooxygenases. Finally, during thelast phase, GA 20-oxidases and GA 3-oxidases catalyzethe last steps of the GA biosynthesis pathway. These are

key enzymes controlling active GA biosynthesis fromGA12 andGA53 (Xu et al., 1995;Williams et al., 1998) thatregulate many developmental processes, such as pro-motion of seed germination. Thus, it has been shownthat GAs and the expression of a GA 20-oxidase(FsGA20ox1) play an important role in breaking thedormancy of beech seeds (Calvo et al., 2004). Also,Perez-Flores et al. (2003) have indicated that the level ofseed dormancy presented by different lines of sorghum(Sorghum bicolor) correlates with the level of expressionof two different GA 20-oxidases. Additionally, it hasbeen shown that GA 3-oxidase genes also play an im-portant role in the stimulation of seed germination inArabidopsis (Yamauchi et al., 2004).

In a previous work, we reported the cloning ofFsPP2C2, encoding a functional PP2C from beechnut,whose expression was induced by ABA in seeds andother tissues (Lorenzo et al., 2002). The alignment of thecatalytic cores of several PP2Cs showed that this PP2Cis included in cluster 3, where PP2Cs have not beenstudied in detail (Saez et al., 2004). The commoncharacteristic of all the phosphatases included in this

Figure 1. Molecular analysis of 35S:FsPP2C2 transgenic lines. A, RNA-blot analysis of transgenic lines overexpressing FsPP2C2. Total RNA(10 mg/line) from wild-type plants and C1 to C3 transgenic plants wasisolated and hybridizedwith a specific FsPP2C2 probe. Bottom, Ethidiumbromide-stained gel showing rRNAs. B, Southern-blot analysis of wild-type (WT; Col-0 background) and transgenic lines (C1–C3) overexpress-ing FsPP2C2. Genomic DNA was digested with HindIII, blotted onto anylon membrane, and hybridized with a FsPP2C2-specific probe.

Figure 2. Effect of FsPP2C overexpression on dormancy and germination in Arabidopsis. A, Dormancy assay of FsPP2C2-overexpressing seeds. Germination percentage was determined 5 d after stratification at 4�C for 0, 24, and 120 h. B, Percentageof seeds that germinated and developed green cotyledons after 8 d following stratification at 4�C for 120 h, in 0.1, 0.2, and 0.3mM

ABA. C, Image showing differences in germination of FsPP2C2-overexpressing, wild-type seeds and seeds transformed with theempty vector pBI121 after 5 d on 0.1 mM ABA. D, Percentage of seeds that germinated and developed green cotyledons on 50 mM

NaCl. E, Percentage of seeds that germinated and developed green cotyledons in 100 mM mannitol. C1, C2, and C3, Transgenic35:FsPP2C2 lines; pBI121, transgenic line with the empty vector pBI121.

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cluster is the presence of two nuclear targeting se-quences next to the C-terminal region of the gene,although there is no experimental evidence of its loca-tion or function. In this work, because there are no toolsavailable to transform beech, we took advantage of theconstitutive expression of FsPP2C2 in Arabidopsis tostudy the function of this protein.

We report here that Arabidopsis plants overexpress-ing FsPP2C2 showed enhanced sensitivity to ABA andabiotic stress and a deeper degree of seed dormancycompared to wild-type seeds and seedlings. Addition-ally, transgenic plants showed a dwarf phenotype,dark-green leaves, and late flowering, these effectsbeing reversed by GA3. We have also confirmed thenuclear localization of this protein phosphatase andfound that ABA is the signal that directs FsPP2C2 to thenucleus. The transgenic lines contain reduced levels ofGAs associated with altered expression of genes in-

volved in the late steps of GA biosynthesis (GA20ox andGA3ox). Taken together, these results are consistent withthe role of FsPP2C2 as a positive regulator of ABAsignaling by inhibiting GA biosynthesis.

RESULTS

Generation and Characterization of Transgenic

Arabidopsis Plants Overexpressing FsPP2C2

To investigate the role of FsPP2C2 in seed dormancyand other ABA responses, we used an overexpressionapproach in Arabidopsis. Transgenic plants were se-lected in the presence of kanamycin. Seventeen ofthem (26%) showed 3:1 segregation for kanamycinresistance in the T2 generation, possibly indicating asingle insertion of the full-length 35S:FsPP2C2 trans-gene, and, finally, three independent T3 homozygous

Figure 3. A, Root growth assay to determine ABA sensitivity. The image was taken 7 d after transfer of 5- to 7-d-old seedlings fromMS medium to plates containing different concentrations of ABA (0, 1, 5, and 10 mM). B, Adult wild-type (left) and FsPP2C2transgenic plants (right). C, Scanning electron microscopic observation of the epidermal cells of the stems. D, Number of rosetteleaves of Col and FsPP2C2-overexpressing lines at the flowering stage both in LD and SD. E, Image showing the size and greeningof 35S:FsPP2C2 and wild-type seedlings.

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lines (from 65 independent lines; C1, C2, and C3)showing high levels of expression of the transgenewere selected (Fig. 1A). Southern-blot analysis of thesehomozygous lines confirmed the presence of a singleinsertion of the 35S:FsPP2C2 transgene (Fig. 1B).

Constitutive Expression of FsPP2C2 in Arabidopsis

Increases Seed Dormancy and Confers Hypersensitivityto ABA and Osmotic Stress

Wild-type and FsPP2C2 transgenic plants wereplated on Murashige and Skoog (MS) media withABA, NaCl, or mannitol included in the media toascertain whether FsPP2C2 overexpression affectedseed germination in response to ABA or osmotic stresstreatment. To determine the degree of dormancy, wecompared germination percentages at 5 d after seedingof 35S:FsPP2C2 and wild-type seeds collected at thesame time after different cold treatment periods (0, 24,and 120 h). The three FsPP2C2 transgenic lines dis-played a greater degree of dormancy compared withwild type after the three kinds of cold treatmentsassayed (Fig. 2A). In the presence of 0.1, 0.2, or 0.3 mM

ABA, germination of FsPP2C2-overexpressing seedswas delayed (Fig. 2, B and C), whereas nearly all wild-type seeds completed germination after 8 d (even with0.3 mM ABA). In contrast, only about 60% and 20% ofFsPP2C2 transgenic seeds completed germination in

the presence of 0.1 and 0.2 mM ABA, respectively, andno radicle protrusion was scored in 0.3 mM ABA. Onthe other hand, no differences in germination percent-ages were observed between wild-type and transgenicseeds in the absence of ABA after 8 d of treatment,although wild-type seeds completed germination to100% at day 6 (data not shown). This delayed com-pletion of germination in FsPP2C2 transgenic seeds,consistent with the deeper degree of dormancy ofthese seeds, may be due to increased sensitivity toendogenous ABA. Thus, all these results indicate thatoverexpression of FsPP2C2 in Arabidopsis confersABA hypersensitivity.

It seems unlikely that these phenotypes were due tomutations caused by random insertion of the trans-gene because many independent transformants wereisolated and around 80% of the transgenic lines ex-hibited the phenotype. In addition, sensitivity to ABAin the pBI121 empty-vector control line did not differsignificantly from those in untransformed wild-typeplants, as shown in Figure 2, B and C, indicating thatthe phenotypes under study are due to the transgeneand not to some peripheral endogenous gene alteredduring transformation.

Germination of FsPP2C2 transgenic seeds was alsosignificantly more sensitive than the wild type to lowconcentrations of NaCl (50 mM; Fig. 2D) and mannitol(100 mM; Fig. 2E). Because it is known that osmotic

Figure 4. Responses of FsPP2C2 transgenic plants to plant hormones. A, Germination percentage of moist, chilled (120 h at 4�C)seeds of wild-type and C1 to C3 transgenic lines 5 d following transfer to 25�C without or with 10 mM GA3. B, Reversion of theinhibitory effect of ABA on germination 10 d after addition of 10 or 100 mMGA3. C, Image showing the stature of wild-type plantsand FsPP2C transgenic plants after the addition of BR (100mM), ACC (100mM), and GA3 (10mM). D, Reversion byGA3 (100mM) oflate-flowering phenotype in LD and SD.

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shock blocks the completion of germination throughABA action (Leung and Giraudat, 1998), these resultsare consistent with the ABA-hypersensitive inhibitionof germination described above.

Constitutive Expression of FsPP2C2 in ArabidopsisConfers Various Phenotypes in Vegetative Tissues

ABA sensitivity in vegetative tissues was also ana-lyzed to determine whether FsPP2C2 overexpressionaffected other plant phenotypes. Thus, comparablewild-type and FsPP2C2 transgenic seedlings grown invertical agar plates lacking ABA were transferred tovertical agar plates supplemented with this hormoneto test the effect of the FsPP2C2 transgene on rootsensitivity to ABA. As seen in Figure 3A, root elonga-tion was significantly more sensitive to ABA in trans-genic plants compared with the wild type.

Additionally, FsPP2C2 transgenic plants showed adwarf phenotype. Adult FsPP2C2-overexpressingplants were approximately one-third as high as thewild type (Fig. 3B), and this dwarf phenotype was dueto smaller length of cells of the stem, as observed byelectron microscopy (Fig. 3C). These transgenic plantsalso exhibited a late-flowering phenotype underboth long-day (LD) and short-day (SD) conditions(Fig. 3D), smaller, dark-green leaves (see Fig. 3E for arepresentative C1 transgenic line; a similar phenotypewas also observed for the other 35S:FsPP2C2 lines),and a different distribution of trichomes (delayedformation of abaxial trichomes and abnormal distri-

bution of adaxial trichomes, just on the edge of the leaf;data not shown). All these features are characteristic ofGA-deficient and GA-insensitive mutants (Koornneefand van der Veen, 1980; Chien and Sussex, 1996;Magome et al., 2004).

Effect of Plant Hormones on 35S:FsPP2C2 Plants

In addition to ABA, the sensitivity of FsPP2C2 trans-genic lines to brassinosteroid (BR), ethylene (using theethylene precursor 1-aminocyclopropane-1-carboxylicacid [ACC]), and GA3 on germination and stem elon-gation was also investigated.

No differences between transgenic and wild-typeplants were observed in response to BR and ACCtreatments (data not shown). However, GA3 was able tocounteract both seed dormancy (Fig. 4A) and seedhypersensitivity to ABA (Fig. 4B) of transgenic linesafter 5 or 10 d of treatment, respectively. Furthermore,GA3 was also able to rescue the short stature of thesetransgenic plants (Fig. 4C) and the late-flowering phe-notype (compare Figs. 4D and 3D). This effect of GA inreversing the phenotypes of 35S:FsPP2C2 suggests thatoverexpression of FsPP2C2 in Arabidopsis plants mightaffect GA biosynthesis in transgenic lines.

Cellular Expression Pattern of FsPP2C2:GreenFluorescent Protein

FsPP2C2, as all the members of cluster 3 of PP2Cs(Saez et al., 2004), has two nuclear targeting sequencesnext to the C-terminal region of the gene product, an

Figure 5. Expression pattern of FsPP2C2 fused to theN-terminal end of GFP in BY2 tobacco cells (A); ABA-untreated onion epidermal cells (D); ABA-treatedonion epidermal cells (E); and 35S:GFP as controls(B and C).

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unusual feature among PP2Cs (Lorenzo et al., 2002).However, no experimental evidence on the subcellularlocalization of this group of PP2Cs has been described.Thus, to verify the possible nuclear localization of thisphosphatase, the FsPP2C2 coding region was fused tothe C- or N-terminal end of the green fluorescentprotein (GFP) under the control of the cauliflowermosaic virus (CaMV) 35S promoter and transientexpression assays were developed in BY2 tobacco(Nicotiana tabacum) cells and in onion (Allium cepa)epidermal cells. The results obtained with the twokinds of constructs were similar, so only picturescorresponding to the construct of the coding regionof FsPP2C2 fused to the N-terminal end of the GFP arepresented (Fig. 5). The results show that, in spite of thetwo nuclear targeting sequences present in FsPP2C2,this protein did not migrate to the nucleus in BY2tobacco (Fig. 5A). This was confirmed in onion epi-dermal cells transformed with FsPP2C2:GFP, where, asin the case of tobacco cells, cytoplasmic distribution ofthe protein was observed in more than 90% of un-treated cells (Fig. 5D). However, ABA applicationinduced migration of FsPP2C2 to the nucleus inmore than 85% of ABA-treated onion cells (Fig. 5E).On the other hand, when cells were treated with GA3,no nuclear localization was observed in any of the cells(data not shown). This indicates the involvement ofFsPP2C2 in ABA signaling because of the absence orpresence of this hormone is a signal that modulates thelocalization of this protein in the nucleus.

Quantification of GAs

Because many of the effects observed in 35S:FsPP2C2 plants were reversed by GAs, we quantifiedthe levels of endogenous GAs in 4-week-old trans-genic and wild-type plants to determine whether GAmetabolism was altered in FsPP2C2-overexpressinglines. As shown in Table I, the levels of the interme-

diate GA19 and the end products GA20 (in the earlyC-13 hydroxylation pathway) and GA9 (in the non-C-13 hydroxylation pathway) of GA 20-oxidase activ-ity were not significantly altered in our transgeniclines with respect to wild-type plants. However, thelevels of GA4 (the main active GA in Arabidopsis) andof GA34 (the inactive metabolite of GA4) were reducedto about 60% in the transgenic lines. Interestingly, thelevels of GA1 and GA8 (the active GA and its inactivemetabolite in the early C-13 hydroxylation pathway)were also reduced in the transgenic lines, as well asGA29 (the inactive metabolite of GA20). These resultsshow that the decrease of bioactive GAs in FsPP2C2transgenic plants was probably due to lower GA3-oxidase activity.

Expression of GA20ox, GA3ox, and a GASA1 Gene

Expression of two genes involved in the last stepsof GA biosynthesis, GA5 (AtGA20ox1, encoding a GA20-oxidase; Xu et al., 1995) and GA4 (AtGA3ox1, en-coding a GA 3-oxidase; Williams et al., 1998), keyenzymes in controlling active GA biosynthesis, werealso analyzed. Transcripts of AtGA20ox1 were notdetected in the wild type but were very abundant intransgenic seedlings (Fig. 6A). Slightly higher levels ofAtGA3ox1 transcripts were also observed in transgenicseedlings, as compared with the wild type (Fig. 6A).Expression of many GA20ox and GA3ox genes indifferent species is subjected to negative feedbackregulation (for review, see Olszewski et al., 2002).Thus, the increase of AtGA20ox1 and AtGA3ox1 tran-scripts found in our transgenic lines is probably aconsequence of the reduction in the level of active GAsdescribed before. To check whether the GA signaltransduction pathway was affected in FsPP2C2-over-expressing lines, we also analyzed the expressionof GASA1, a GA-induced gene (Herzog et al., 1995),in both GA-treated and untreated transgenic and

Figure 6. A, Expression of AtGA20ox1 (GA5) andAtGA3ox1 (GA4) genes. B, GASA1 gene expressionin FsPP2C2-overexpressing plants (C1 and C2) com-pared to wild type. mRNA levels of the indicatedgenes were determined by semiquantitative RT-PCR(A) or northern-blot analysis (B) using total RNAs(10 mg/line) isolated from 7-d-old seedlings. Bottom,Ubiquitin (A) and ethidium bromide-stained gelshowing rRNAs (B) as controls.

Table I. GA amount (ng g21 fresh weight) in Arabidopsis plants from wild-type and FsPP2C2 transgenic lines (C1 and C2)

Levels of active GAs (GA1 and GA4) are in bold. Values are means of three biological replicates6SE, except in the case of GA8 (only two replicates).

GA19 GA20 GA29 GA1 GA8 GA9 GA4 GA34

Wild type 0.47 6 0.13 0.23 6 0.03 0.20 6 0.07 1.06 6 0.26 1.00 0.22 6 0.07 1.25 6 0.18 0.68 6 0.14C1 0.51 6 0.11 0.17 6 0.03 0.09 6 0.07 0.83 6 0.04 0.08 0.29 6 0.10 0.71 6 0.10 0.43 6 0.10C2 0.40 6 0.12 0.13 6 0.02 0.04 6 0.02 0.45 6 0.14 0.10 0.15 6 0.07 0.73 6 0.13 0.42 6 0.06

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wild-type plants. As seen in Figure 6B, expression ofthis gene is up-regulated in our transgenic lines afterGA treatment, although such up-regulation was not soclear in wild-type plants, as previously described(Herzog et al., 1995; Aubert et al., 1998). This resultindicates that the mechanism of GA action is notaffected in transgenic lines overexpressing FsPP2C2.

Expression of an ABA-Responsive Gene

We compared the expression of the RAB18 gene, aknown ABA-responsive gene (Lang and Palva, 1992)in 35S:FsPP2C2 plants, with that of Columbia (Col-0)to examine whether the enhanced ABA sensitivity intransgenic plants was accompanied by altered expres-sion of ABA-responsive genes. The hyperinduction ofthis ABA-responsive gene further confirms that thePP2C from beech is involved in ABA signal transduc-tion. RAB18 transcripts were not detected in wild-typeand untreated transgenic lines (Fig. 7, lanes 1–3), butwere induced by ABA treatment, much more so intransgenic that in wild-type lines (Fig. 7, lanes 4–6).

DISCUSSION

Only six PP2Cs are found in the yeast (Saccharomycescerevisiae) genome (Stark, 1996) and no more than 15 inthe human genome (Cheng et al., 2000). In contrast, thehigh number of PP2C genes (at least 69) present inthe Arabidopsis genome denotes the importance ofthe PP2C class of Ser-Thr phosphatases in plants (Kerket al., 2002) and suggests that plant PP2Cs have anarrower substrate specificity than other organisms(Merlot et al., 2001).

One plant PP2C family (cluster 5; Saez et al., 2004) hasbeen identified as constituted by components of ABAsignaling. For instance, ABI1, ABI2, AtPP2CA, HAB1,and FsPP2C1 act as negative regulators of the ABAsignal transduction cascade (Gosti et al., 1999; Merlotet al., 2001; Tahtiharju and Palva, 2001; Gonzalez-Garcıaet al., 2003; Saez et al., 2004; Kuhn et al., 2006; Yoshidaet al., 2006). Other members of PP2C, such as MP2C(cluster 6; Saez et al., 2004) from ice plant (Mesembry-anthemum crystallinum), act as negative regulators of themitogen-activated protein kinase pathway activated bysalt stress or wounding (Meskiene et al., 1998, 2003).

Interestingly, all these PP2Cs act as negative regulatorsin different signal transduction cascades. In fact, exceptfor the recent controversy on the role of ABI1 from thework of Wu et al. (2003), all the PP2Cs described andcharacterized so far would act as negative regulators indifferent signal transduction cascades.

We have previously isolated FsPP2C2, a PP2C frombeech that is up-regulated by ABA in seeds andvegetative tissues (Lorenzo et al., 2002), making it areasonable candidate as a regulator of ABA signaling.However, genetic evidence of the role of this phos-phatase was lacking. Studies of orthologous genes andfunctional tests in heterologous systems have shownthat the ABA signal transduction cascade is essentiallyconserved among evolutionarily distant plant species(for review, see Finkelstein et al., 2002). Therefore, inthis study, we used an overexpression approach inArabidopsis to investigate FsPP2C2 function. It wasexpected that if FsPP2C2 is a negative regulator ofABA responses, as is another PP2C described in beech(FsPP2C1; Lorenzo et al., 2001; Gonzalez-Garcıa et al.,2003), the overexpression of FsPP2C2 in Arabidopsiswould lead to ABA insensitivity in seeds and, conse-quently, to reduced dormancy. Surprisingly, 35S:FsPP2C2plants exhibited an enhanced response to ABA andosmotic stress (Figs. 2 and 3) together with otherphenotypes, such as dwarfism, late flowering, smallerand dark-green leaves (Fig. 3), and abnormal trichomedistribution (data not shown), all these typical

Figure 8. Proposed model for the role of FsPP2C2 in ABA signalingthrough a reduction of GA biosynthesis.

Figure 7. Expression of the RAB18 ABA-regulated gene in FsPP2C2-overexpressing plants (C1 and C2) compared to wild type. mRNA levelsof the indicated genes were determined by northern-blot analysis usingtotal RNAs (10 mg/line) isolated from mock-treated (2) or ABA-treated(150 mM ABA for 24 h) plants. Bottom, Ethidium bromide-stained gelshowing rRNAs.

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characteristics of GA-deficient and/or insensitive mu-tants (Koornneef and van der Veen, 1980; Magomeet al., 2004). Furthermore, these phenotypes, includ-ing enhanced response to ABA, were reversed by GA3application (Fig. 4).In addition to the genes ABI1, ABI2, PP2CA, HAB1,

and FsPP2C1 (Gosti et al., 1999; Merlot et al., 2001;Tahtiharju and Palva, 2001; Gonzalez-Garcıa et al.,2003; Saez et al., 2004; Kuhn et al., 2006; Yoshida et al.,2006) encoding PP2C, other genes have been identifiedas negative regulators of the ABA-signaling pathway,such as the farnesyl transferase b-subunit (ERA1;Cutler et al., 1996), the mRNA cap-binding protein(ABH1; Hugouvieux et al., 2001), the homeodomainprotein 6 (ATHB6; Himmelbach et al., 2002), and thecalcium sensor CBL-9 (Pandey et al., 2004). Moreover,ABI3, ABI4, and ABI5 encode different types of tran-scription factors (Giraudat et al., 1992; Finkelstein et al.,1998; Finkelstein and Lynch, 2000) known to act aspositive regulators of ABA-mediated regulation ofseed development, germination, and early seedlinggrowth (Finkelstein et al., 2002; Arroyo et al., 2003).However, FsPP2C2 is described as a positive regulatorof the ABA signal transduction pathway in seeds andother vegetative tissues. Evidence of the role of thisPP2C on ABA action was obtained from the observa-tion that plants overexpressing FsPP2C2 had hyper-sensitivity to ABA and osmotic stress and a deeperdegree of dormancy compared with wild-type plants(Figs. 2 and 3). Expression of the ABA-responsive geneRAB18 was also increased in 35S:FsPP2C2 plants (Fig.7). Moreover, the fact that FsPP2C2 is localized in thenucleus only in the presence of ABA (Fig. 5) stronglysupports its involvement in ABA signaling.Evidence of the mode of action of FsPP2C2 in ABA

responses was initially obtained from the observationthat GA3 was able to rescue the poor germination,dwarfism, and delay in flowering of transgenic plantsoverexpressing FsPP2C2 (Fig. 4). Because GA actiondid not seem to be affected in these plants (Fig. 6B),this indicated that FsPP2C2 might exert its action bymodulating GA metabolism. Quantification of endog-enous GAs showed that the levels of active GAs (GA4and GA1) were significantly reduced in transgeniclines, whereas the contents of their immediate meta-bolic precursors (GA9 and GA20, respectively) were notaffected (Table I). The low reduction of GA4 content(about 50%) in transgenic lines displaying phenotypeagrees with the low increase of active GA4 (2- to 3-fold)in transgenic Arabidopsis overexpressing GA20ox.This is in contrast with the 10-fold increases in appliedGA3 needed to get partial reversal of germinationphenotypes in the presence of ABA (Fig. 4B). However,this is similar to what happens in pea (Pisum sativum)ovary, where small differences in active GA content(within 1 order of magnitude) have very significantphysiological effects, whereas higher doses of GA1(orders of magnitude) have to be applied exogenouslyto produce a similar effect probably due to transportand/or metabolism problems (Rodrigo et al., 1997).

Our results suggest that overexpression of FsPP2C2decreased GA 3-oxidase activity. The lower content ofinactive metabolites of GA4 and GA1 (GA34 and GA8,respectively) also supports this conclusion, whileshowing that the decrease of active GA content wasnot the result of higher inactivating metabolism. In-terestingly, transcript levels of AtGA3ox1 (gene GA4)were not reduced, but rather increased in transgeniclines (Fig. 6A). An even more dramatic effect wasobserved on AtGA20ox1 (gene GA5) transcript levels(Fig. 6A). It is known that expression of many GA20oxand GA3ox genes is controlled by active GAs throughnegative feedback regulation (Olszewski et al., 2002).Therefore, the increase of GA5 and GA4 transcripts isprobably a result of this kind of regulation and we canpostulate that the regulation of GA 3-oxidase activityby FsPP2C2 is probably carried out at the posttransla-tional level.

In conclusion, the results presented in this work areconsistent with the role of the ABA-induced FsPP2C2as a positive regulator of ABA signaling through areduction of GA biosynthesis, probably affecting GA3-oxidase activity (Fig. 8). In this proposed model, wehypothesize that a phosphorylated protein in thenucleus may activate an unknown element responsiblefor the activation of GA 3-oxidase, but ABA actionthrough FsPP2C2 would dephosphorylate this pro-tein, leading to inactivation of this key enzyme in GAbiosynthesis. Thus, a potential interaction betweenABA action and GA biosynthesis is described, show-ing another junction in the complex mechanism ofhormone signaling. The data presents new insightsinto future research about the antagonistic effect ofABA and GAs in plant development.

MATERIALS AND METHODS

Plant Materials

Arabidopsis (Arabidopsis thaliana) plants, ecotype Col-0, were used in this

research. They were normally grown in a growth chamber with 40% humidity,

at 22�C, under SD (8 h light/16 h dark) or LD (16 h light/8 h dark; light

intensity of 80–100 mE m22 s21) in pots containing a 1:3 vermiculite:soil

mixture, as described previously (Gonzalez-Garcıa et al., 2003). For in vitro

culture, seeds were surface sterilized in 70% (v/v) ethanol solution containing

1% (v/v) Triton X-100 and washed four times in sterile distilled water.

Stratification of seeds was conducted during 3 d at 4�C, unless otherwise

indicated. Afterward, seeds were sowed on MS plates (Murashige and Skoog,

1962) containing solid medium composed of MS basal salts and 1% (w/v) Suc,

pH 5.7, solidified with 1% (w/v) agar. Plates were sealed and incubated in a

controlled-environment growth chamber.

Vector Construction and Plant Transformation

To produce transgenic plants, the coding region of the FsPP2C2 cDNAwas

cloned into the pBIN121 vector, which contains the modified CaMV 35S

promoter. 5#-GGATCCATGTTTTCGGA-3# (sense) and 5#- GAGCTCTTAGG-

TGCTGCCAAC-3# (antisense), containing the BamHI and SacI cloning sites,

were used as primers to amplify FsPP2C2 cDNA and subcloned in the

corresponding sites of the pBIN121 vector. The pBIN121-FsPP2C2 construct,

after confirmation of the nucleotide sequence, was introduced into Agro-

bacterium tumefaciens C58C1 (pGV2260; Deblaere et al., 1985) by heat shock.

Arabidopsis plants (Col-0 ecotype) were transformed by the floral-dip method

(Clough and Bent, 1998) and transgenic seedlings were selected on kanamycin

PP2C Related to Abscisic Acid Responses and GA Biosynthesis

Plant Physiol. Vol. 141, 2006 1421

medium (50 mg mL21). T2 plants that produced 100% kanamycin-resistant

plants in the T3 generation were considered homozygous for the selection

marker and used for further studies. Nontransformed plants and transgenic

plants transformed with the empty vector pBI121 were used as controls.

From 65 kanamycin-resistant independent lines rescued, around 80%

showed an ABA-hypersensitive phenotype in seed germination compared

with controls that did not display any phenotypes related to ABA responses.

Germination Assays

Seeds were plated on solid medium composed of MS basal salts, 1% (w/v)

Suc, and different concentrations of ABA (Sigma-Aldrich; 0.1, 0.2, 0.3 mM) to

determine ABA sensitivity. For the dormancy assay, seed lots to be compared

were harvested on the same day from individual plants grown in identical

conditions and were stratified during 0, 1, and 5 d at 4�C.To determine sensitivity to inhibition of germination by osmotic stress, the

medium was complemented with low concentrations of mannitol (100 mM) or

NaCl (50 mM).

Reversion of the inhibitory effect of ABA in seed germination was carried

out using media containing 10 and 100 mM GA3 (Sigma-Aldrich).

Seed germination was scored by determining daily the percentage of seeds

that had germinated and developed green cotyledons for 5 to10 d. At least

three replicates of each germination assay were performed.

Root Growth and Flowering Time

Root growth assay for scoring ABA sensitivity was carried out by mea-

suring root growth 7 d after transferring 5- to 7-d-old seedlings onto vertical

MS plates containing different concentrations of ABA (0, 1, 5, and 10 mM). The

leaf number used to determine flowering phenotype was performed as

described by Wilson et al. (1992).

Hormone Dose Response Experiments

Ten-day-old seedlings were transferred to pots containing a 1:3 vermiculite:

soil mixture. The seedlings were sprayed every 2 d with GA3 (100 mM), BR

(Epibrassinolide; 10 mM), or ACC (100 mM; Sigma-Aldrich).

Northern-Blot Analysis

Total RNA was extracted using the RNA wiz kit (Ambion) following the

manufacturer’s protocol, separated on formaldehyde-agarose gels, and blot-

ted onto a nylon membrane. Blots were hybridized with [32P]-labeled specific

probes. All RNA gel-blot experiments were repeated at least three times, and

results from one representative experiment are shown in the figures. RAB18

and GASA1 probes were prepared by reverse transcription (RT)-PCR with the

primers described elsewhere (Herzog et al., 1995; Gonzalez-Guzman et al.,

2002). Blots were exposed for 24 h in a phosphor imager screen (Fujitsu).

Semiquantitative RT-PCR

cDNA was synthesized from total RNA using the first-strand cDNA

synthesis kit for RT-PCR of Avian myeloblastosis virus (Roche Diagnostics) with

oligo-p(dT) as primer, following the manufacturer’s instruction. Two micro-

liters were used for each PCR consisting of 1-min cycles at 94�C, 50�C, and72�C. AtGA20ox1, AtGA3ox1, and UBQ14 primers were those used by Oka

et al. (2001), Williams et al. (1998), and Vriezen et al. (2004), respectively. Each

PCR was performed for 15, 20, 25, and 30 cycles following the procedure

described by Vriezen et al. (2004). After Southern blotting and hybridizing

with the purified PCR products, the signals were quantified by phosphor

imager analysis (Fujitsu). The amplification reactions were exponential be-

tween 15 and 25 cycles, and the products of the reactions after 20 cycles were

used for quantification. The experiment was performed twice and produced

reproducible patterns.

FsPP2C2:GFP Constructs

The FsPP2C2 coding region was fused to the N-terminal end of GFP in the

vector pMON30063 (Pang et al., 1996), under the control of the CaMV 35S

promoter. A fragment corresponding to the FsPP2C2 coding region was ampli-

fied by PCR using synthetic anchored primers that contained aNcoI-compatible

restriction site at their 3# and 5# ends. In addition, the coding region of FsPP2C2

was also fused to the C-terminal end of GFP in the same vector, although in this

case the subsequent cloning required a different strategy. The pMON vector was

digested with EcoRI restriction enzyme, whereas FsPP2C2 was digested with

NdeI and XhoI restriction enzymes because its coding region contained an EcoRI

restriction site. Both the linearized vector and the DNA fragment containing

FsPP2C2 coding regions were treated with the Klenow fragment of DNA

polymerase in order to obtain blunt ends before ligation. Following sequence

verification of the inserts, GFP fusion and control constructs were transiently

expressed by particle bombardment.

Transient Expression Assays

Transformation was carried out following the procedure described by

Varagona et al. (1992). DNA absorption to gold particles and bombardment

using a helium-driven particle accelerator (PDS-1000/He; Bio-Rad) was

performed according to the manufacturer’s recommendations. One micro-

gram of plasmid was used for transformation, and the target material (BY2

tobacco [Nicotiana tabacum] and onion [Allium cepa] epidermal cells) were

bombarded three times. After bombardment, onion epidermal cells were

transferred to MS medium supplemented or not with 100 mM ABA and kept at

28�C in the dark for 18 h before analysis by fluorescence microscopy. Cells

were examined using a Zeiss Axiovert 200 microscope equipped with

epifluorescence optics. GFP-specific fluorescence was detected using Zeiss

filters 515 to 530 nM. Confocal images were obtained on a Zeiss LSM 510

confocal laser-scanning microscope with Zeiss LSM version 2.8 software.

Scanning Electron Microscopy

Stems were harvested from mature plants and observed directly by a

scanning microscope (Zeiss DSM 940) equipped with a cooling stage.

Quantification of GAs

Endogenous GAs were quantified by gas chromatography-mass spectrom-

etry as described elsewhere (Garcıa-Martınez et al., 1997). The material (about

10 g fresh weight of rosette leaves from 4-week-old plants grown at 22�Cunder LD conditions, at the vegetative state, and collected 3 h after starting

the light period) was spiked at the time of extraction with [3H]GA20 (1.41

TBq mmol21) and [3H]GA9 (1.41 TBq mmol21) to check recoveries during the

purification procedure, and with [17-2H2]GA1, [17-2H2]GA4, [17-2H2]GA8,

[17-2H2]GA9, [17-2H2]GA19, [17-2H2]GA20, [17-2H2]GA29, and [17-2H2]GA34

(purchased from Prof. L. Mander, Research School of Chemistry, Australian

National University, Canberra, Australia) as internal standards for quantifi-

cation. Fractions of HPLC containing the GAs of interest were methylated and

trimethylsilylated before analysis by gas chromatography-mass spectrometry

using a Hewlett-Packard 5890 gas chromatograph coupled to a 5971A mass

selective detector. The peak-area ratios of the following ion pairs, in the

appropriate HPLC fractions and having a retention time similar to that of the

corresponding GAs, were determined to calculate the concentrations of

endogenous GAs by reference to calibration curves: 506/508 (GA1), 284/286

(GA4), 594/596 (GA8), 298/300 (GA9), 434/436 (GA19), 418/420 (GA20), 506/

508 (GA29), and 506/508 (GA34).

We only measured GA amounts in the transgenic C1 and C2 lines because

the size and production of seeds of the C3 line were not sufficient to perform

this set of experiments.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession number AJ277744.

ACKNOWLEDGMENTS

We thank Dr. P.L. Rodriguez (Instituto de Biologıa Molecular y Celular de

Plantas-Consejo Superior de Investigaciones Cientificas, Valencia), for

providing us with Arabidopsis seeds transformed with the empty vector

pBI121, used as a control in this study.

Received June 8, 2006; revised June 8, 2006; accepted June 21, 2006; published

June 30, 2006.

Reyes et al.

1422 Plant Physiol. Vol. 141, 2006

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