The Magnesium-Chelatase H Subunit Binds Abscisic …...cytokinin-like regulator 6-benzylaminopurine,...

The Magnesium-Chelatase H Subunit Binds AbscisicAcid and Functions in Abscisic Acid Signaling:New Evidence in Arabidopsis1[W][OA]

Fu-Qing Wu2, Qi Xin2, Zheng Cao2, Zhi-Qiang Liu2, Shu-Yuan Du, Chao Mei, Chen-Xi Zhao,Xiao-Fang Wang, Yi Shang, Tao Jiang, Xiao-Feng Zhang, Lu Yan, Rui Zhao, Zi-Ning Cui, Rui Liu,Hai-Li Sun, Xin-Ling Yang, Zhen Su, and Da-Peng Zhang*

State Key Laboratory of Plant Physiology and Biochemistry (F.-Q.W., Q.X., Z.C., Z.-Q.L., X.-F.W., T.J., L.Y.,R.Z., R.L., H.-L.S., Z.S.) and College of Science (Z.-N.C., X.-L.Y.), China Agricultural University, Beijing100094, China; and Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084,China (S.-Y.D., C.M., C.-X.Z., Y.S., X.-F.Z., D.-P.Z.)

Using a newly developed abscisic acid (ABA)-affinity chromatography technique, we showed that the magnesium-chelatase Hsubunit ABAR/CHLH (for putative abscisic acid receptor/chelatase H subunit) specifically binds ABA through the C-terminalhalf but not the N-terminal half. A set of potential agonists/antagonists to ABA, including 2-trans,4-trans-ABA, gibberellin,cytokinin-like regulator 6-benzylaminopurine, auxin indole-3-acetic acid, auxin-like substance naphthalene acetic acid, andjasmonic acid methyl ester, did not bind ABAR/CHLH. A C-terminal C370 truncated ABAR with 369 amino acid residues(631–999) was shown to bind ABA, which may be a core of the ABA-binding domain in the C-terminal half. Consistently,expression of the ABAR/CHLH C-terminal half truncated proteins fused with green fluorescent protein (GFP) in wild-typeplants conferred ABA hypersensitivity in all major ABA responses, including seed germination, postgermination growth, andstomatal movement, and the expression of the same truncated proteins fused with GFP in an ABA-insensitive cchmutant of theABAR/CHLH gene restored the ABA sensitivity of the mutant in all of the ABA responses. However, the effect of expression ofthe ABAR N-terminal half fused with GFP in the wild-type plants was limited to seedling growth, and the restoring effectof the ABA sensitivity of the cch mutant was limited to seed germination. In addition, we identified two new mutant alleles ofABAR/CHLH from the mutant pool in the Arabidopsis Biological Resource Center via Arabidopsis (Arabidopsis thaliana)Targeting-Induced Local Lesions in Genomes. The abar-2 mutant has a point mutation resulting in the N-terminal Leu-348/Phe, and the abar-3 mutant has a point mutation resulting in the N-terminal Ser-183/Phe. The two mutants show alteredABA-related phenotypes in seed germination and postgermination growth but not in stomatal movement. These findingssupport the idea that ABAR/CHLH is an ABA receptor and reveal that the C-terminal half of ABAR/CHLH plays a centralrole in ABA signaling, which is consistent with its ABA-binding ability, but the N-terminal half is also functionally required,likely through a regulatory action on the C-terminal half.

The phytohormone abscisic acid (ABA) regulatesmany aspects of plant growth and development, suchas seed maturation, germination, and seedling growth,and is a central hormone in the control of plantadaptation to environmental challenges, includingdrought, salt, and cold stresses, by regulating stomatalaperture and expression of stress-responsive genes(for review, see Koornneef et al., 1998; Leung and

Giraudat, 1998; Finkelstein and Rock, 2002). ABAfunctions through a complex network of signalingpathways, where ABA signal perception by ABAreceptors is the primary event that triggers down-stream signaling cascades to induce the final physio-logical responses. Numerous cellular components thatmodulate ABA responses downstream of ABA recep-tors have been identified (for review, see Finkelsteinet al., 2002; Himmelbach et al., 2003; Fan et al., 2004),leading to considerable progress in understandingABA signaling pathways. ABA signal perception,which has been considered to be mediated by multiplereceptors, including plasma membrane and intracel-lular receptors (for review, see Assmann, 1994;Finkelstein et al., 2002), has attracted much attention.In recent years, two classes of plasma membrane ABAreceptors, an unconventional G-protein-coupled re-ceptor (GPCR), GCR2, and a novel class of GPCR,GTG1 and GTG2, that regulate the major ABA re-sponses in seed germination, seedling growth, andstomatal movement (Johnston et al., 2007; Liu et al.,

1 This work was supported by the National Natural ScienceFoundation of China (grant no. 90817104 to D.-P.Z. and grant no.30700053 to X.-F.W.).

2 These authors contributed equally to the article.* Corresponding author; e-mail [email protected] 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:Da-Peng Zhang ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.109.140731

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2007a, 2007b; Pandey et al., 2009), have been reported.However, whether GCR2 regulates ABA-mediatedseed germination and postgermination growth is con-troversial, because the ABA-related phenotypes areweak to absent in gcr2 mutants (Gao et al., 2007; Guoet al., 2008). GTGs are positive regulators of ABAsignaling and interact with the sole Arabidopsis (Arabi-dopsis thaliana) G-protein a-subunit, GPA1, that maynegatively regulate ABA signaling by inhibiting theactivity of GTG-ABA binding (Pandey et al., 2009).Most recently, a PYR/PYL/RCAR family of STARTproteins was reported to function as a cytosolic ABAreceptor by inhibiting directly type 2C protein phos-phatases, which is suggested to trigger a reversibleprotein phosphorylation cascade to mediate ABA re-sponses (Ma et al., 2009; Park et al., 2009).We previously reported the magnesium-proto-

porphyrin IX (ProtoIX) chelatase large subunit (Mg-chelatase H subunit; CHLH) as an ABA receptorresiding in chloroplasts and mediating ABA responsesin seed germination, postgermination growth, andstomatal movement (Shen et al., 2006). CHLH hasmultiple functions in plant cells. As a subunit of theMg-chelatase, CHLH catalyzes the introduction of Mgto ProtoIX, a key regulatory step of chlorophyll bio-synthesis. In addition, CHLH plays a key role in me-diating plastid-to-nucleus retrograde signaling (forreview, see Nott et al., 2006). We found that a broadbean (Vicia faba) ABA-specific binding protein, ABAR(for putative abscisic acid receptor), is encoded by theCHLH gene (Zhang et al., 2002; Shen et al., 2006). In thereference plant Arabidopsis, we showed that ABAR/CHLH specifically binds ABA and mediates ABAsignaling as a positive regulator, indicating thatABAR is an intracellular ABA receptor. We showedalso that ABAR-mediated ABA signaling is distinctfrom chlorophyll biosynthesis and plastid-to-nucleusretrograde signaling (Shen et al., 2006). However, inbarley (Hordeum vulgare), a recent report showed thatthe barley chlorophyll-deficient mutants with muta-tions in the XanF gene (the same large subunit of Mg-chelatase as the CHLH in Arabidopsis) showed noABA-related phenotypes in seed germination, post-germination growth, and stomatal movement, and theABA-binding activity of XanF was not detected usingthe system of the authors, which led them to questionthe ABA-receptor nature of ABAR/CHLH, at least inbarley (Muller and Hansson, 2009). Here, we report,using a newly developed ABA-affinity chromatogra-phy technique and a [3H]ABA-binding assay, thatABAR/CHLH was shown to specifically bind ABAvia the C-terminal half but not the N-terminal half.Expression of the ABAR C-terminal half truncatedprotein in wild-type plants and in an ABA-insensitivecch mutant of ABAR/CHLH induced much strongerABA-related phenotypes than expression of theN-terminal half truncated protein did, revealing a crit-ical role of the C-terminal half in ABA signaling. Char-acterization of two new mutant alleles with pointmutations in the N-terminal half of ABAR/CHLH,

abar-2 and abar-3, suggests that the N-terminal halfmay cover a regulatory domain involved in modulatingC-terminal function. These data support the idea thatthe Mg-chelatase H subunit is an ABA receptor.

RESULTS

Affinity Chromatography Shows That ABARSpecifically Binds ABA

We developed an ABA-affinity chromatographytechnique to detect ABA binding to a known proteinessentially based on our ABA-binding protein purifi-cation procedures (Zhang et al., 2002). An ABA-linkedSepharose 4B was used to assess qualitatively whethera given protein binds ABA. The Escherichia coli-expressed full-length ABAR was loaded onto theABA-linked Sepharose 4B column and first eluted by150 mM NaCl of total volume 36 mL at 3 mL each for 12times (Fig. 1A, lanes 1–12) to eliminate completelyunspecifically bound or loosely bound protein by thecolumn. It is noteworthy that this eliminated portionby the 150 mM NaCl elution should also include adisassociated portion of an ABA-specific binding pro-tein in the reversible binding process. After this elu-tion, the column was further eluted by 100 or 150 mM

NaCl plus 4 mM (6)ABA. This ABA-affinity compet-itive elution released efficiently the ABA-specific bind-ing portion of the ABAR protein (Fig. 1A, lanes 13–15).In contrast, elution with the inactive isomer 2-trans,4-trans-ABA could not substantially recover the ABARprotein (Fig. 1B, lanes 13–15). As a negative control, theSepharose 4B column that was not coupled with ABAdid not bind tightly to the ABAR protein. The looselybound protein was completely removed from the non-ABA-linked columnwith 150mMNaCl of total volumeabout 15 mL at 3 mL each for five times (Fig. 1C, lanes1–5), and a subsequent elution with 100 mM NaCl plus4 mM (6)ABA did not recover any detectable ABARprotein (Fig. 1C, lanes 13–15). It is noteworthy that theprotein eluted by 150 mM NaCl from the ABA-linkedcolumn needed, in most cases, about 30 mL of elutionwith 3 mL each for nine to 10 times (Fig. 1, A and B,lanes 1–10), showing that the ABA-linked columnbound the ABAR protein specifically and tightly. The4 mM ABA elution with either 100 or 150 mM NaClshowed no difference in efficiency in recovering ABARprotein from the ABA-affinity column (Fig. 1, A andD), indicating that this ABA-specific elution was in-dependent of NaCl concentrations in the range from100 to 150 mM.

To further test the specificity of binding of ABARprotein to the ABA-affinity column, the ABAR proteinwas incubated with 50 or 500 nM (+)ABA in order tocompete with the Sepharose 4B-linked ABA for free(+)ABA. The ABA concentration of 50 nM is slightlylower than the saturation concentration of ABA bind-ing to ABAR protein (disassociation constant [Kd] = 32nM; Shen et al., 2006; Fig. 1, J and K). The concentration

ABAR/CHLH Mediates ABA Signaling

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Figure 1. Affinity chromatography shows that ABAR binds ABAvia the C-terminal half but not the N-terminal half. A, The E. coli-expressed, purified, full-length ABAR was loaded onto an ABA-linked Sepharose 4B chromatography column and eluted by 150mM NaCl of total volume 36 mL at 3 mL each for 12 times (lanes 1–12) to remove unspecifically bound or loosely bound proteinby the column. The eluate was subjected to 10% SDS-PAGE, and the ABAR protein was detected by the anti-full-length ABARserum with immunoblotting. Note that the protein eluted by 150 mM NaCl from the ABA-linked column needed, in most cases,about 30 mL of elution with 3 mL each for nine to 10 times (lanes 1–10). The column was eluted, after the elution with 150 mM

NaCl, by 100 mM NaCl plus 4 mM (6)ABA, which released the ABA-specific binding portion of the ABAR protein (lanes 13–15).The left panel shows molecular mass markers (kD). B, The ABA-specific binding portion of the ABAR protein could not be elutedsubstantially from the column by the ABA isomer 2-trans,4-trans-ABA (lanes 13–15) under the same elution conditions as used inA: only a trace of the ABAR protein was detected (lane 13). C, A negative control. The Sepharose 4B column that was not coupledwith ABA did not tightly bind the ABAR protein. The loosely bound protein was completely removed from the non-ABA-linkedcolumn with 150 mM NaCl of total volume about 15 mL at 3 mL each for five times (lanes 1–5), and a subsequent elution with100 mM NaCl plus 4 mM (6)ABA did not recover any detectable ABAR protein (lanes 13–15). D, The same assays were performedas in A, but for the ABA-specific elution the combination of 150 mM NaCl plus 4 mM (6)ABA was used instead of 100 mM NaClplus 4 mM (6)ABA. Note that the same result was observed as in A. E, Competition assays of ABA binding to the ABA-affinitycolumn. The protein sample was preincubated with 0, 50, or 500 nM (+)ABA (top panels) before being loaded onto the column,

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of the Sepharose 4B-linked (6)ABAwas about 500 nM

in the binding-incubation system (see “Materials andMethods”), equivalent to 250 nM (+)ABA. The incuba-tion of ABAR protein with 50 nM free (+)ABA effi-ciently decreased the amount of ABAR protein elutedfrom the column, and the immunosignal of the ABARprotein was undetectable with the 500 nM (+)ABAincubation (Fig. 1E). This competition assay showedthat the ABAR binding to the ABA-affinity column isspecific. The competition efficiency of the free (+)ABAwith Sepharose-bound ABA for ABAR protein washigh, likely because the important carboxyl group atthe C1 site of ABA is blocked by the Sepharosecoupling, which may decrease the ability of bindingto ABAR protein in comparison with free (+)ABA.In addition to trans-ABA, we used a set of potential

agonists/antagonists to ABA, gibberellin GA3, cytokinin-like regulator 6-benzylaminopurine (6-BA), auxinindole-3-acetic acid (IAA), and jasmonic acid methylester (MeJA), to elute ABAR protein from the ABA-affinity column in place of (6)ABA. However, elutionwith any of these substances did not recover detectableABAR protein (Fig. 1F, lanes 13–15), showing that thesesubstances are not able to bind the ABAR protein.To get further evidence that the Arabidopsis natural

ABAR binds ABA, the Arabidopsis total protein wasloaded on the ABA-linked Sepharose 4B, and thecolumn was subjected to the same elution procedures

mentioned above. The ABA-affinity competitive elu-tion recovered efficiently the ABAR protein from theABA-linked Sepharose 4B column but not from theABA-free column (Fig. 1G). A competition assay,performed with the same procedure as described forthe E. coli-expressed ABAR protein (Fig. 1E), showedthat preincubation of the Arabidopsis total proteinwith 50 nM (+)ABA decreased significantly the amountof ABAR protein eluted from the column, and the 500nM (+)ABA preincubation completely eliminated theABAR immunosignal (Fig. 1G). These results, consis-tent with those of E. coli-expressed ABAR protein,showed that the Arabidopsis natural ABAR protein isable to bind ABA.

Additionally, using this ABA-affinity column, weshowed that the E. coli-expressed barley XanF bindsABA and that, in the barley natural proteins, both thefull-length and a truncated XanF (if it exists naturally)bindABA (Supplemental Fig. S2). In rice, the twonaturalCHLHs both bind ABA (Supplemental Figs. S2 and S3).

The C-Terminal Half of ABAR Binds ABA, But theN-Terminal Half Does Not

To assess ABA-binding domains in the ABAR mol-ecule, three truncated ABAR proteins were used toperform the ABA-affinity chromatography assays. Thetruncated C-terminal C751 protein (amino acid resi-

Figure 1. (Continued.)and the columnwas subjected to the same elution procedures as in D. Note that the 50 nM ABA treatment decreased significantlythe amount of ABAR protein eluted from the column, and the immunosignal of the ABAR protein was undetectable with the 500nM ABA treatment. The protein samples displayed in lanes 13, 14, and 15 [ABAR protein eluted by 150 mM NaCl plus 4 mM

(6)ABA] were further assayed by immunoblotting on the same gel (bottom panel, red arrow), and the relative amounts of theimmunodetected ABAR protein were estimated by scanning the bands with the values given above the bands. F, The potentialagonists/antagonists, other phytohormones, or plant growth regulators GA3, cytokinin-like regulator 6-BA, auxin IAA, and MeJAare not able to bind the ABAR protein. The ABA-linked Sepharose 4B affinity column was eluted by 100 mM NaCl plus 4 mM ofthe substances used separately after 150 mM NaCl elution of 36 mL at 3 mL each for 12 times under the same conditions as in A.Elution with any of these substances did not recover detectable ABAR protein (lanes 13–15). The symbol X indicates the differentsubstances. G, The Arabidopsis natural ABAR protein binds ABA. The Arabidopsis total protein was loaded on the ABA-linkedSepharose 4B, and the column was subjected to the same elution procedures as described in A. The 100 mM NaCl plus 4 mM

(6)ABA elution after the 150 mM NaCl elution recovered the ABAR protein from the ABA-linked column (top panel, Sepharose4B-ABA) but not from the ABA-free Sepharose 4B column (Sepharose 4B). An incubation of the protein sample with 50 nM(+)ABA (50 nM ABA/Sepharose 4B-ABA) decreased significantly the ABAR protein bound to the ABA-affinity column, andincubation with 500 nM (+)ABA completely abolished the ABAR binding to the column: no immunosignal of ABAR protein wasdetected with the ABA-specific elution (500 nM ABA/Sepharose 4B-ABA). A control was performed with 0 nM (+)ABA but thesame amount of ethanol for solubilizing 500 nM ABA (0 nM ABA/Sepharose 4B-ABA). Lanes 1, 2, and 3 denote immunosignals ofthe first and last elution with 150 mM NaCl and the first elution with 100 mM NaCl plus 4 mM (6)ABA, respectively. H, Thetruncated C-terminal C751 protein (amino acid residues 631–1,381; a) and C370 protein (amino acid residues 631–999; b) bindABA, but the N-terminal truncated N772 protein (amino acid residues 1–772; c) does not. The C751, C370, and N772 proteinswere loaded on the ABA-linked Sepharose 4B column that was subjected to the same elution procedures as in A. The 100 mM

NaCl plus 4 mM (6)ABA elution after the 150mMNaCl elution recovered the C751 (a) and C370 (b) proteins from the ABA-linkedcolumn (lanes 13 and 14), but the N772 protein was removed quickly from the column by 150 mM NaCl elution (c) and was notrecovered by the 100 mM NaCl plus 4 mM (6)ABA elution (lanes 13 and 14). I, The C751 truncated protein was not recoveredfrom the ABA-linked column by an elution with 100 mM NaCl plus 4 mM IAA-like substance NAA (lanes 13 and 14). The elutionprocedures were the same as in H. All of the experiments shown here were replicated three times with similar results. J, SaturableABA-specific binding to the pure E. coli-expressed full-length ABAR and truncated C751 and C370 proteins. Asterisks indicatesignificant differences at P , 0.05 (Student’s test) when comparing values (means 6 SD, n = 3) within the same [3H]ABAconcentration. The arrow indicates the unspecific binding data for the three proteins, which are all lower than 10% of totalbinding. K, Scatchard plot of binding data in J. The parameters of the curves are as follows: for ABAR, Kd = 32 nM, Bmax = 1.27 molmol21, r2 = 0.97; for C751, Kd = 40 nM, Bmax = 1.09 mol mol21, r2 = 0.96; for C370, Kd = 41 nM, Bmax = 0.89 mol mol21, r2 = 0.96.





dues 631–1,381; Fig. 2A), and a fragment located in theC751 portion, C370 truncated protein (amino acidresidues 631–999; Fig. 2A), were shown to bind ABA(Fig. 1H, a and b). The specificity of ABA binding tothe C751 truncated protein was assayed using a set ofpotential agonists/antagonists (including all of thesubstances used above and additionally the auxin-likesubstance naphthalene acetic acid [NAA]) as forABAR full-length protein (Fig. 1F), and the resultsshowed that these potential agonists/antagonists werenot able to compete with ABA for binding to the C751truncated protein, revealing that the C751-ABA bind-ing is specific (Fig. 1I; data not shown). However, theN-terminal N772 truncated protein (amino acid resi-dues 1–772; Fig. 2A) was removed quickly from theABA-linked column by 150 mM NaCl elution, of whichthe immunoblotting band disappeared almost com-pletely in the fourth elution (total volume, 12 mL) by150 mM NaCl (Fig. 1H, c, lanes 1–4), while the immu-noblotting bands of the C751 and C370 truncatedproteins were still retained in the ninth or 10th elutionby 150 mM NaCl (total volume, 27–30 mL; Fig. 1H, aand b, lanes 9 and 10). Importantly, the N772 truncatedprotein was not recovered by the ABA-affinity com-petitive elution (Fig. 1H, c, lanes 13 and 14). All ofthese findings showed that the N-terminal N772 trun-cated protein did not bind ABA.

The ABA-binding parameters of the C751 and C370truncated ABAR proteins were assayed by a [3H]ABAin-buffer binding system. The C751 truncated proteinbinds ABAwith a Kd of 40 nM and maximum binding(Bmax) of 1.09 mol ABA mol21 protein, and the C370truncated protein binds ABA with a Kd of 41 nM andBmax of 0.89 mol mol21 (Fig. 1, J and K; SupplementalTable S1). The mutations of C751 and C370 affected thetwo ABA-binding parameters compared with the full-length ABAR (Kd = 32 nM, Bmax = 1.27 mol mol21; Shenet al., 2006), but both the truncated ABAR proteinscould still perceive ABA at the nanomolar level.

Expression of the C-Terminal Half of ABAR in the

Wild-Type Plants Confers ABA Hypersensitivity in Allof the Major ABA Responses, But the Effect ofExpression of the N-Terminal Half Was Limited to

Seedling Growth

To test the functional domains of ABAR/CHLHbased on the findings from the ABA-binding assays,we first created transgenic lines in the wild-typeecotype Columbia (Col-0) background using four dif-ferent constructs of ABAR/CHLH cDNA fragments.We previously found that the expression of the full-length ABAR open reading frame often caused cosup-pression of the ABAR/CHLH gene, which made itdifficult to obtain transgenic lines. This was alsoobserved by another group (Strand et al., 2003). Wehypothesized that there might be a sequence respon-sible for the protein/mRNA stability of ABAR/CHLHand tried to use a truncated ABARwith the C-terminal78 amino acid residues deleted (amino acid residues

1–1,303, called ABARn; Fig. 2A), which indeed made itmuch easier to obtain transgenic lines (see below). Wealso tried to use a truncated ABAR with N-terminaldeletion of 100 to 200 amino acid residues covering thechloroplast transit peptide, but we failed to obtaintransgenic lines; therefore, we used an N-terminal 310-amino acid-deleted ABAR truncation (amino acidresidues 311–1,381, named ABARc) for transgenicmanipulation (Fig. 2A). The other three constructsincluded the truncated ABARs used in the above-mentioned ABA-binding assays (i.e. C751, C370[linked to the N-terminal chloroplast transit peptide,amino acid residues 1–120], and N772; Fig. 2A). Thesetruncated ABARs were fused with GFP and shown tocorrectly express in the transgenic lines (Fig. 2B) andlocalized in chloroplasts except for ABARc and C751,which localized in the cytosol because of lack of thechloroplast transit peptide (Fig. 2D). Expression of anyof the truncated ABARs in the wild-type backgrounddid not affect the contents of Mg-ProtoIX, ProtoIX, andchlorophyll of the transgenic lines, showing that thechlorophyll biosynthesis was not altered substantially(Fig. 2, E and F). In contrast, the ABA sensitivity ofthese transgenic plants was changed (Figs. 3–5). Thetransgenic plants expressing the four truncatedABARs ABARn, ABARc, C751, and C370 all showedsignificantly the ABA-hypersensitive phenotypes inseed germination, postgermination growth (Fig. 3),and ABA-induced stomatal closure and ABA-inhibitedstomatal opening (Fig. 5A). However, the trans-genic plants expressing the N-terminal N772 truncatedprotein showed substantially wild-type phenotypes inseed germination (Fig. 3A) and in postgerminationgrowth when the seeds were planted directly in theABA-containing medium (Fig. 3B), while the ABA-hypersensitivity phenotype in seedling growth wasobserved in the N772 transgenic plants when theseedlings were transferred to the ABA-containingmedium from the ABA-free medium 48 h after strati-fication (Fig. 3, C andD), which suggests the complexityof the mechanisms involved in the ABAR-mediatedABA signaling. In ABA-induced stomatal closure andABA-inhibited stomatal opening, the N772 transgenicplants showed wild-type phenotypes (Fig. 5A). It isnoteworthy that the transformation of the plants withempty vector (control vector harboring GFP) did notinduce any chlorophyll- or ABA-related phenotypes(Supplemental Fig. S1).

Expression of the C-Terminal Half of ABAR in the

ABA-Insensitive cch Mutant Restores ABA Sensitivity inAll of the Major ABA Responses, But the Effect ofExpression of the N-Terminal Half Was Limited toSeed Germination

We created transgenic lines in the ABA-insensitivecch mutant background using the same constructs ofABAR/CHLH cDNA fragments as in wild-type Col-0.Molecular analysis showed that the constructs werecorrectly expressed in the transgenic plants without

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Figure 2. Molecular, cellular, and biochemical analysis of the ABAR cDNA fragment transgenic plants. A, Diagram showing thetruncated ABAR expressed in the transgenic lines. ABARn and ABARc, The truncated ABAR with the C-terminal 1,304-to-1,381sequence (78 amino acid residues) and the N-terminal 1-to-310 sequence (310 amino acid residues) deleted, respectively. ABAR,Full-length ABAR. The cch mutation site in the ABAR protein is indicated. C751, The truncated ABAR with the N-terminal 1-to-630 sequence deleted. C370, The truncated ABAR with the N-terminal 121-to-630 sequence and the C-terminal 1,000-to-1,381sequence deleted but fused with the 1-to-120 sequence covering the transit peptide. N772, The ABAR N-terminal 1-to-772sequence only. B, Immunoblot analysis of the transgenic lines with both the anti-full-length ABAR and anti-Actin sera. The leftpanels show the molecular mass markers (kD). Col, Wild-type plants; 1, 2, and 3 indicate three independent transgenic lines. Inall panels, ABAR indicates the natural ABAR protein and Actin indicates the loading control. a, ABARn transgenic lines. ABARnindicates the GFP-tagged ABARn transgenic protein. b, ABARc transgenic lines. ABARc indicates the GFP-tagged ABARctransgenic protein. c, C751 transgenic lines. C751 indicates the GFP-tagged C751 transgenic protein. d, C370 transgenic lines.C370 indicates the transgenic protein of the GFP-tagged C370 plus the N terminal 1-to-120 sequence. e, N772 transgenic lines.





altering the cch mutation (Fig. 2C; data not shown).Expression of two truncated ABARs, ABARn andC370, linked to the chloroplast transit peptide en-hanced the contents of Mg-ProtoIX and ProtoIX of thetransgenic lines, although the contents were still lowerthan in the wild-type plants (Fig. 2E). However, thechlorophyll contents of the transgenic lines were com-parable to those in the wild-type plants (Fig. 2F).Contrarily, expression of the other three truncated

proteins, ABARc, C751, and N772, did not restore thelevels of Mg-ProtoIX, ProtoIX, and chlorophyll (Fig. 2,E and F). For the ABA-related phenotypes, expressionof C751 and N772 truncated ABAR restored the ABAsensitivity in seed germination of the cch mutant,while expression of the ABARn, ABARc, and C370truncated proteins made the cch transgenic seedshypersensitive to ABA compared with the wild-typeplants (Fig. 4A). However, the expression of the N772

Figure 2. (Continued.)N772 indicates the GFP-tagged N772 transgenic protein. C, Molecular analysis of the cch mutant transgenic lines. A 257-bpPCR-amplified fragment with an introduced HaeIII restriction site can be cut into a smaller sequence for the wild-type ABAR(ABAR-f) but cannot be cut for the cch mutation (cch-f), so that the transgenic lines in the cch mutant background harbor twobands of the PCR products. Col, Wild-type plants; cch, cch mutant; ABARn, ABARc, C370, N772, and C752 indicate ABARn,ABARc, C370,N772, and C751 transgenic lines, respectively. D, Transient expression of the GFP-tagged ABARn, ABARc, C751,C370, and N772 constructs in Arabidopsis protoplasts. GFP, Fluorescence of the GFP-tagged truncated ABAR protein; Auto-,chlorophyll autofluorescence; Bright, bright field; Merged, overlay of the three images. Note that ABARn was predominantlylocalized to the envelope of chloroplasts, ABARc and C751 were localized to cytosol, and C370 and N772 were localized to thechloroplast stroma. E, The concentrations of Mg-ProtoIX and ProtoIX in leaves of the transgenic plants. F, The concentrations ofchlorophyll in leaves of the transgenic plants. Chla, Chlb, and Total indicate chlorophyll a, chlorophyll b, and total amounts oftwo kinds of chlorophyll, respectively. Each value in E and F is the mean 6 SE of three biological determinations, and differentletters indicate significant differences at P, 0.05 (Student’s test) when comparing values within the same group. Other symbolsin E and F are as in C.

Figure 3. Phenotypic analysis of trans-genic lines of the wild-type back-ground in seed germination andpostgermination growth. A, Seed ger-mination rate of the wild-type plants(Col) and different transgenic lines inthe ABA-free medium (0 mM ABA) andABA-containing medium (0.5 mM) from24 to 60 h after stratification. B, Post-germination growth of the wild-typeplants (Col) and different transgeniclines in the ABA-free medium (0 mM

ABA) and 0.5 mM ABA-containing me-dium 12 d after stratification. The seedswere directly planted in the ABA-freeor ABA-containing medium to investi-gate the response to ABA after germi-nation. C and D, Seedling growth ofthe wild-type plants (Col) and differenttransgenic lines in the ABA-free me-dium (0 mM ABA) and ABA-containingmedium 12 d after seedling transfer.Seeds were germinated after stratifica-tion on common MS medium andabout 48 h later transferred to theABA-containing medium in the verti-cal position. Seedling growth was in-vestigated 12 d after the transfer (D),and the length of primary roots wasmeasured (C). Each value in A and C isthe mean 6 SE of three biological de-terminations, and asterisks indicatesignificant differences at P , 0.05(Student’s test) when comparing valueswithin the same point of time (A) or thesame ABA concentration (C).

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truncated ABAR had no effect on postgerminationgrowth and stomatal movement, while the expressionof the other four truncated ABARs, ABARn, ABARc,C751, and C370, restored the ABA sensitivity in both ofthe ABA major responses (Figs. 4, B–D, and 5B). It isnoteworthy that the transformation of the cch mutantplants with empty vector (a control) did not induceany phenotypic change (Supplemental Fig. S1).

Characterization of Two Mutant Alleles in the ABARGene Reveals That the N-Terminal Half Is Required to

Regulate ABA Signaling

We identified two new mutant alleles of the ABAR/CHLH gene, abar-2 and abar-3, from the mutant pool inthe Arabidopsis Biological Resource Center (ABRC)via the Arabidopsis Targeting Induced Local Lesionsin Genomes (TILLING) project (Henikoff et al., 2004).The abar-2 mutation had a C-to-T change at nucleotide1,042, and abar-3 had a C-to-T substitution at nucleo-tide 548, resulting in Leu-348/Phe and Ser-183/Phe

mutations, respectively, in ABAR protein (Fig. 6A),both of which localize in the N-terminal half of theABAR/CHLH protein. Neither of the mutationsaffects Mg-ProtoIX and chlorophyll production (Fig.6, B and C), but both of the mutations alter theABA sensitivity in germination and postgerminationgrowth (Fig. 6, D–G). The abar-2 mutant has an ABA-insensitive phenotype in seed germination, like the cchmutant (Fig. 6D), while the abar-3mutant has an ABA-hypersensitive phenotype in seed germination (Fig.6D), contrary to abar-2. The abar-3 plants showed astronger ABA-insensitive phenotype in postgermina-tion growth, like the cch mutant, when the seeds wereplanted directly in the ABA-containing medium (Fig.6, E–G), but the abar-2 plants showed a weaker ABA-insensitive phenotype in postgermination growth onlywhen postgermination growthwas prolonged (Fig. 6E;1 mM ABA with seedling growth for 30 d). However,both abar mutants have substantially no ABA-relatedphenotypes in ABA-induced stomatal closure andABA-inhibited stomatal opening (data not shown).

Figure 4. Phenotypic analysis of cch mutant trans-genic lines in seed germination and postgerminationgrowth. A, Seed germination rate of wild-type (Col)and cch mutant plants and different transgenic linesin the ABA-free medium (0 mM ABA) and ABA-containing medium (0.5 mM) from 24 to 60 h afterstratification. B, Postgermination growth of cch mu-tant plants (cch) and different cch mutant transgeniclines in the ABA-free medium (0 mM ABA) and 0.5 mM

ABA-containing medium 7 d after stratification. Theseeds were directly planted in the ABA-free or ABA-containing medium to investigate the response toABA after germination. C and D, Seedling growth ofwild-type (Col) and cch mutant plants and differenttransgenic lines in the ABA-free medium (0 mM ABA)and ABA-containing medium 14 d after seedlingtransfer. Seeds were germinated after stratificationon common MS medium and about 48 h latertransferred to the ABA-containing medium in thevertical position. Seedling growth was investigated14 d after the transfer (D), and the length of primaryroots was measured (C). Each value in A and C is themean 6 SE of three biological determinations, andasterisks indicate significant differences at P , 0.05(Student’s test) when comparing values within thesame point of time (A) or the same ABA concentra-tion (C).





DISCUSSION

ABA-Affinity Chromatography Reveals That ABARBinds ABA via the C-Terminal Half But Not theN-Terminal Half

The newly developed ABA-affinity chromatogra-phy technique allowed us to assay the ABAR/CHLHABA binding with a different system from those weused previously (Shen et al., 2006) and further to assess

the ABA-binding domain in this protein. Unspecificelution by 150 mM NaCl showed that the ABA-affinitycolumn bound tightly the ABAR/CHLH protein com-pared with the Sepharose 4B column uncoupled withABA, but it bound the non-ABA-binding ABAR trun-cated N772 protein loosely (Fig. 1). The longer retainedABAR/CHLH protein in the ABA-affinity columnduring the unspecific elution process (Fig. 1) maylikely be an ABA-specific binding portion of ABAR/CHLH that was reversibly disassociated from theaffinity column during the known receptor-ligandreversible binding process. Importantly, the tightlyand specifically bound portion of ABAR/CHLH to theABA-affinity columnwas efficiently eluted by (6)ABAbut not by a set of potential agonists/antagonists,including 2-trans,4-trans-ABA, GA3, 6-BA, IAA, NAA,and MeJA (Fig. 1). Furthermore, this ABA-affinitycolumn allowed us to pull down the natural ABAR/CHLH protein, but the Sepharose 4B column uncou-pled with ABA could not (Fig. 1). Free (+)ABA at 50 nM

competed efficiently with the Sepharose-linked ABAfor both E. coli-expressed and natural ABAR protein(Fig. 1), indicating that ABAR bound ABA at thenanomolar level. These findings showed that theABA-affinity chromatography technique is specificand reliable for detecting ABA-binding proteins, onthe one hand, and that ABAR/CHLH directly bindsABA specifically, on the other hand. Using this ABA-affinity column, we showed that the C-terminal half ofABAR/CHLH, but not the N-terminal half, binds ABA(Fig. 1). A C-terminal C370 truncated ABAR with 369amino acid residues (631–999) was shown to bindABA, which may be a core of the ABA-binding do-main in theC-terminal half.With the in-buffer [3H]ABA-binding assays we conducted previously (Shen et al.,2006), we further showed that the two C-terminaltruncated ABAR proteins bound ABA at the nano-molar level, which allows plant cells to perceive phys-iological concentrations of ABA, although theirABA-binding ability decreases compared with full-length ABAR (Fig. 1).

Using both the in-buffer radiolabeled ABA-bindingsystem and a pull-down assay, we previously showedthat ABAR/CHLH binds ABAwith high affinity (Kd =32 nM), high stereospecificity [with (–)ABA and trans-ABA unable to compete with (+)ABA in the ABAbinding], and in a saturable, reversible manner andthat an ABAR/CHLH protein can bindmaximally 1.28ABA molecules (Shen et al., 2006), which meets theprimary criteria for receptor-ligand binding. Consis-tently, this experiment, using a newly developed sys-tem, provides, to our knowledge, new evidence thatABAR/CHLH is an ABA-specific binding protein.

The C-Terminal Half of ABAR Plays a Central Role inABA Signaling, and the N-Terminal Half Is AlsoFunctionally Required

The transgenic manipulation showed that expres-sion of the three C-terminal truncated ABARs, ABARc,

Figure 5. Phenotypic analysis of the transgenic plants in ABA-inducedstomatal closure and ABA-inhibited stomatal opening. A, ABA-inducedstomatal closure (top) and inhibition of stomatal opening (bottom) intransgenic plants of the wild-type background. B, ABA-induced sto-matal closure (top) and inhibition of stomatal opening (bottom) in cchmutant transgenic plants. Values are means 6 SE from three indepen-dent experiments, and different letters indicate significant differences atP, 0.05 (Student’s test) when comparing values within the same group(within the same ABA concentration applied for assaying stomatalaperture); n = 60 apertures per experiment.

Wu et al.




Figure 6. Characterization of two new mutant alleles, abar-2 and abar-3, in the ABAR/CHLH gene and a hypothetical model forABAR function. A, Diagram showing the locations of the abar-2 and abar-3mutations in the ABAR genomic DNA. B and C, Theconcentrations of Mg-ProtoIX and ProtoIX (B) as well as chlorophyll a (Chl a), chlorophyll b (Chl b), and total chlorophyll (TotalChl; C) in wild-type plants (Col) and cch, abar-2, and abar-3 mutants. D, Seed germination rates of the wild-type plants and thethree mutants in the medium supplemented with different concentrations of (6)ABA (0, 0.5, 1, and 3 mM) from 24 to 84 h afterstratification. E, Postgermination growth of the wild-type plants (Col) and the three mutants in the medium supplemented with0.5, 1, or 3 mM (6)ABA at different periods (14, 17, or 20 d) after stratification. Seeds were directly planted in the ABA-containingmedium to investigate the response of seedling growth to ABA after germination. F and G, Seedling growth (estimated by rootlength) of the wild-type plants (Col) and the threemutants in the ABA-free medium (0mM ABA) and ABA-containingmedium (0.5,1, and 3 mM) 30 d after stratification. Seeds were directly planted on plates containing medium supplemented with ABA, which





C751, and C370, in wild-type Col-0 plants conferredABA hypersensitivity and, in the ABA-insensitive cchmutant, restored ABA sensitivity in all of the ABAresponses, including ABA-inhibited seed germination,postgermination growth, ABA-induced stomatal clo-sure, and ABA-inhibited stomatal opening (Figs. 3–5).The effects induced by expression of these three trun-cated ABARswere comparable to those induced by thesubstantially full-length ABAR (ABARn; Figs. 3–5),although the ABARc and C751 truncated proteinsuncoupled with the chloroplast transit peptide wereexpressed in cytosol but not in chloroplast (Fig. 1). Allof these findings provide evidence for a central roleof the C-terminal half of ABAR/CHLH in ABA sig-naling.

The underlying mechanism of ABAR-mediatedABA signaling across the chloroplast envelope re-mains an open question. The ABARc and C751 trans-genically induced effects suggest that the C-terminalhalf of ABAR/CHLH may function in cytosol tomediate ABA signaling. The function of the truncatedABARs in the cytosolic side allows a hypotheticalmodel that the entire ABAR molecule may move,through the chloroplast envelope, into the cytosolicspace in response to ABA stimulation or that, struc-turally, ABARmay have transmembrane domains (e.g.C or N terminus) across the chloroplast envelope intothe cytosolic side, with which ABAR mediates cyto-solic signaling events. The work to test this hypothesisis ongoing in our laboratory. A recent report showedthat a chloroplast protein, NRIP1, is recruited to thecytoplasm and nucleus by the tobacco mosaic virusp50 effector to mediate pathogen recognition pro-cesses in plant cells (Caplan et al., 2008), supportingthe idea of chloroplast protein trafficking through thechloroplast envelope.

It is noteworthy that the expression of the fourtruncated ABARs (ABARn, ABARc, C751, and C370)in the wild-type plants did not significantly change thechlorophyll biosynthesis when altering ABA re-sponses, and the expression of the ABARc and C751truncated proteins uncoupled with the chloroplasttransit peptide in the cch mutant affected ABA re-sponses but did not restore the low chlorophyll level ofthe yellow mutant, which reveals that the ABAR/CHLH-mediated ABA signaling is distinct from chlo-rophyll biosynthesis, consistent with our previousobservation (Shen et al., 2006). It is also noteworthythat the C370 truncated protein linked to the N-terminalchloroplast transit peptide restored chlorophyll biosyn-thesis in the cch mutant as the substantially full-length ABAR (ABARn) did (Fig. 2), suggesting that

this C-terminal fragment is crucial to both ABA signal-ing and chlorophyll biosynthesis.

The expression of the N-terminal truncated ABARN772 protein in the wild-type plants did not affect,and in the cch mutant did not restore, chlorophyllbiosynthesis. However, the N772 expression partiallyaltered the ABA-responsive phenotypes in seedlinggrowth of the wild-type plants and in seed germina-tion of the cch mutant (Figs. 3 and 4) but not instomatal response to ABA in either transgenic wild-type or cch mutant plants (Fig. 5). Two mutant alleleswith point mutations in the N-terminal half of ABAR/CHLH, abar-2 and abar-3 (Fig. 6), have chlorophylllevels comparable to the wild-type plants but signif-icantly altered ABA responses in seed germinationand postgermination growth (Fig. 6), whereas neitherof the two point mutations significantly affects stoma-tal responses to ABA (data not shown), contrary to thecch mutant, which has a strong ABA-insensitive phe-notype in stomatal movement (Shen et al., 2006; Fig. 5).These findings support the idea that the N-terminalhalf of ABAR/CHLH plays a secondary role com-pared with the C-terminal half, which is consistentwith the findings that the N-terminal half does notbind ABA (Fig. 1) but the N-terminal half is stillfunctionally required, at least partly, in the modulationof ABA signaling. Distinct from its regulatory role inABA signaling, the N-terminal half may have nofunction, or at least no important function, in chloro-phyll biosynthesis, consistent once again with the ideathat the ABAR-mediated signaling is independent ofchlorophyll biosynthesis.

Wepreviously showed that down- andup-regulationof ABAR/CHLH expression resulted in strong ABAinsensitivity and hypersensitivity, respectively, in all ofthe major ABA responses in Arabidopsis (Shen et al.,2006). The experiment described here provides new,additional evidence for the involvement of ABAR/CHLH in ABA signaling through both transgenic ma-nipulation and creation of new point mutations in theABAR/CHLH gene via TILLING. Taken together withthe ABA-binding data both previously reported (Shenet al., 2006) and presented in this experiment (Fig. 1), allof the findings support the idea that ABAR/CHLH isan intracellular receptor for ABA.

In this regard, however, we understand the currentcontroversy over the ABA receptor nature of ABAR/CHLH. Although all of the data we have for ABAR/CHLH to date are consistent with the essential criteriaof an ABA receptor, there still exist a series of crucialquestions to be answered in relation to the ABAreceptor identity of ABAR/CHLH. For example, al-

Figure 6. (Continued.)were held in the vertical position. Root growth was investigated 30 d after stratification (F), and the length of primary roots wasmeasured (G). Each value in B to D andG is the mean6 SE of three biological determinations, and different letters (in B, C, andG)or asterisks (in D) indicate significant differences at P , 0.05 (Student’s test) when comparing values within the same group ofvalues. H, A hypothetical model of ABAR functional domains. The length of each domain is an approximate estimation. See“Discussion” for explanation.

Wu et al.




though ProtoIX was previously shown to be unable tocompete with ABA for binding to ABAR/CHLH (Shenet al., 2006), does ABA partly share a binding site withProtoIX in the ABAR/CHLH molecule because thestructure of ABA is similar to that of ProtoIX, whichalso binds CHLH? This possible unspecific binding,even if trace, would lead to an overestimation orunderestimation of the number of ABA-binding sites.The most important open question is how ABAR/CHLH transmits ABA signal to downstream regula-tors to mediate the ABA response, particularly if orhow it could transduce the signal to genetically de-fined, currently well-characterized ABA signaling reg-ulators. To answer these questions is essential to definea bona fide ABA receptor.Nevertheless, the experimental data obtained here

allow us to postulate a working model of the func-tional domains in the ABAR/CHLH molecule in theregulation of ABA signaling (Fig. 6H). The C-terminalhalf is likely to play central role in ABA signal per-ception and signal output, covering the ABA-bindingdomain and possibly the domains interacting withdownstream players to relay the ABA signal. Theextreme C terminus is most likely to be a domainresponsible for protein/mRNA degradation. TheN-terminal half may cover, in addition to a transitpeptide, a regulatory center that may be involved inregulation of the functions of the C-terminal half. Totest this postulation in the future will provide newinsight into the ABAR-mediated ABA perception anddownstream signaling.

Is the Large Subunit of Mg-Chelatase Also a CandidateReceptor for ABA in Monocotyledonous Plants?

In contrast to the previous report, in which the ABA-binding activity of the barley XanF was not detected(Muller and Hansson, 2009), ABA-affinity chromatog-raphy allowed us to reveal that both rice (Oryza sativa)CHLH (OsCHLH) and barley XanF are ABA-bindingproteins (Supplemental Figs. S2 and S3).The ability of ABA binding to XanF and OsCHLH

suggests that the two proteins may be involved inABA signaling, although the determination of theABA-binding kinetics will be necessary with an in-buffer radiolabeled ABA-binding system optimizedfor barley and rice to assess if the binding propertiesmeet the essential criteria of ligand-receptor binding.Genetic approaches including transgenic manipula-tions will aid in testing if the two large subunits of Mg-chelatase XanF and OsCHLHmodulate ABA signalingin monocotyledonous plants. First, however, it is note-worthy that a possible functional redundancy of XanFgenes in barley and OsCHLH in rice may occur, whichis different from Arabidopsis, which harbors a singlecopy of CHLH. Rice has two copies of the CHLH gene,one coding for a full-length CHLH and another for aC-terminal truncated CHLH (Supplemental Fig. S3).We showed that the two OsCHLH proteins both bindABA and uncovered the possibility that barley may

have also two XanFs, as in rice (Supplemental Fig. S2).The Arabidopsis C-terminal half of ABAR/CHLHbinds ABA and regulates ABA signaling (Figs. 1–6),which suggests the potential ABA signaling function-ality of the smaller CHLH in rice and also possibly inbarley. In this case, mutations in only one copy of theCHLH/XanF genes may not be able to alter ABAresponsibility. Second, as it was observed in Arabi-dopsis that the ABAR/CHLH-mediated ABA signal-ing is independent of chlorophyll biosynthesis, thechlorophyll-deficient mutants do not necessarily havedefects in ABA responses. This may be one possibleexplanation for why the two chlorophyll-deficientmutants of the barley XanF gene showed wild-typeABA responses (Muller and Hansson, 2009). Third, weobserved that the ABA-insensitive phenotypes of theRNA interference (RNAi) lines of the ABAR/CHLHgene became weaker when the mRNA of ABAR/CHLHdecreased to a very low level (in the RNAi mutantswith pale yellow leaves), suggesting that a strongfeedback effect is involved in the ABAR/CHLH-mediated ABA signaling (Shen et al., 2006). This maybe explained by a possible up-regulation of other ABAsignaling pathways (mediated by other receptors forABA) when the ABAR/CHLH-mediated signalingpathway is down-regulated to a certain low thresholdlevel. Stomatal response, however, did not show thislow-threshold-related phenomenon and displayed astrong ABA insensitivity that was negatively corre-lated with the ABAR/CHLH mRNA level in theseABAR/CHLH-RNAi lines (Shen et al., 2006). So themutants of barley with low XanF protein and paleleaves may be subjected to a similar feedback phe-nomenon as in the severe ABAR/CHLH-RNAi lines ofArabidopsis. Lastly, we do not exclude the possibilitythat CHLH/XanF may not function in ABA signalingin monocotyledonous plants, probably due to theoccurrence of possibly different signaling networksbetween the monocotyledonous and dicotyledonousplants. An RNAi manipulation resulting in down-regulation of the full-length CHLH/XanF and smallerCHLH/XanF may be necessary in barley and rice,respectively, and will aid in clarifying whether the twoproteins are involved in ABA signaling.

MATERIALS AND METHODS

Plant Materials and Generation of Transgenic Plants

Arabidopsis (Arabidopsis thaliana Col-0) and the cch mutant of the ABAR/

CHLH gene were used in the generation of truncated ABAR/CHLH transgenic

plants. Truncated ABAR genes were amplified by PCR from Col-0 cDNAwith

KOD-plus DNA polymerase (Toyabo) and cloned into the binary vector

pCAMBIA1300, which contains the cauliflower mosaic virus 35S promoter

and a C-terminal GFP flag. The ABAR cDNA fragments were isolated by

PCR using the forward primer 5#-GGACTAGTATGGCTTCGCTTGTGTATT-

CTC-3# and reverse primer 5#-GGGGTACCACTCCATCCCACAGTGTT-

GGA-3# for the ABARn fragment (corresponding to the 1–1,303 amino acid

residue-encoding cDNA sequence); the forward primer 5#-GGACTAGTATG-

GACACCAATGACTCACTCAAG-3# and reverse primer 5#-GGGGTACCTC-

GATCGATCCCTTCGATCTTG-3# for the ABARc fragment (corresponding to

the 311–1,381 amino acid residue-encoding cDNA sequence); the forward





primer 5#-GGACTAGTATGGCTTCGCTTGTGTATTCTC-3# and reverse

primer 5#-GGGGTACCATCAAGATTACATTGCTTAGC-3# for the N772 frag-

ment (corresponding to the 1–772 amino acid residue-encoding cDNA

sequence); the forward primer 5#-GGACTAGTATGCCCATGAGGCT-

GCTTTTCTCC-3# and reverse primer 5#-GGGGTACCTCGATCGATCCC-

TTCGATCTTG-3# for the C751 fragment (corresponding to the 631–1,381

amino acid residue-encoding cDNA sequence); and the forward primer

5#-GGACTAGTATGCCCATGAGGCTGCTTTTCTCC-3# and reverse primer

5#-GGGGTACCTGTTGTGGGAATAGCCTGAGG-3# for the C370 fragment

(corresponding to the 631–999 amino acid residue-encoding cDNA sequence).

The C370 fragment was then ligated with a chloroplast transit peptide

fragment that was isolated using the forward primer 5#-GGACTAGTATGG-

CTTCGCTTGTGTATTCTC-3# and reverse primer 5#-CGGGGCCCTCTA-

AGCTCCTCGACCAAGTA-3#. All constructs were confirmed by sequencing.

These constructs were introduced into the GV3101 strain of Agrobacterium

tumefaciens and transformed into plants by floral infiltration. The homozygous

T3 seeds of the transgenic plants were used for analysis. At least five transgenic

lines were obtained for each construct, and all of the lines had similar ABA-

related phenotypes. The results from one representative line (or three in some

cases, as indicated) are presented in this report. The cch mutant was a generous

gift from Dr. J. Chory (Salk Institute). The seeds of the abar-2 and abar-3mutants

were obtained from the ABRC as mentioned below. All of the mutants were

isolated from ecotype Col-0.

Plants were grown in a growth chamber at 19�C to 20�C on Murashige and

Skoog (MS) medium (Sigma) at about 80 mmol photons m22 s21 or in compost

soil at about 120 mmol photons m22 s21 over a 16-h photoperiod. It is

particularly noteworthy that the cch mutant was originally identified as a

light-sensitive mutant deficient in chlorophyll (Mochizuki et al., 2001) and

later shown to be insensitive to ABA in stomatal movement (Shen et al., 2006).

So the cchmutant is more susceptible to both strong light and water deficiency

than its wild-type background Col-0, and mild-stress conditions to Col-0 are

probably severe to the cchmutant. We observed that the good growth status of

the cch mutant parental plants is of critical importance to their progeny to

display the ABA-related phenotypes: the stressed mutant parental plants had

progeny with seeds whose insensitive phenotypes to ABA became weaker in

germination and postgermination growth, but the ABA-insensitive phenotype

in stomatal movement was not affected. This may be due to a possible up-

regulation of ABA-responsive mechanisms independent of ABAR-mediated

signaling, which may be induced by environmental stresses imposed on this

mutant.

Identification of the cch Mutation in the cch Background

Transgenic Lines

The single-nucleotide cch mutation in the cch background transgenic lines

was identified according to a procedure described previously (Neff et al.,

1998). A 257-bp fragment was amplified by PCR, and a HaeIII restriction site

was introduced into the wild-type sequence corresponding to the cchmutation

site, while the cch mutation in the fragment results in abolishment of this

restriction site. The forward primer used was 5#-AGGCTGCTTTTCTC-

CAAGTCAGCAAGGC-3# and the reverse primer was 5#-TTGGCATAA-

CTTCTCCTCTTTG-3#.

Identification of Two ABAR Mutants, abar-2 and abar-3,from TILLING Lines

The abar-2 and abar-3 mutants were identified via the Arabidopsis TILL-

ING project (Henikoff et al., 2004). The tilled M3 seeds with predicted

deleterious mutations in the ABAR/CHLH gene were provided by the ABRC.

Two lines, CS89100 and CS92346 (ABRC stock numbers), were identified as

the abar alleles with the altered phenotypes of ABA sensitivity and are

designated abar-2 and abar-3, respectively. CS89100 (abar-2) had a C-to-T

change at nucleotide 1,042. CS92346 (abar-3) had a C-to-T substitution at

nucleotide 548. Homozygous mutant plants were identified by PCR using

allele-specific primers designed as described by Konieczny and Ausubel

(1993) via restriction site analysis. A 544-bp fragment was amplified by PCR

using the forward primer 5#-GCTTGTTAGGACTTTGCCTAAG-3# and re-

verse primer 5#-TCCACCAACAAGAGCAAAAC-3# for analyzing the abar-2

mutant. An AluI restriction site is present in the wild-type fragment that can

be degraded into 150- and 394-bp fragments, while the abar-2 mutation

abolished this restriction site in the fragment that cannot be cut. Similarly,

a 330-bp fragment was amplified using the forward primer 5#-GAG-

GAATTGGCGATTAAAGT-3# and reverse primer 5#-CTGAAGATTATCAG-

GAGAGCCTC-3# for analyzing the abar-3 mutant. An MboI restriction site is

present in the wild-type fragment that can be cut into 109- and 221-bp

fragments, while this restriction site was abolished by the abar-3mutation. The

two alleles were back-crossed three times to remove the erecta allele that was

present in the parental background and additional mutations induced by

ethyl methanesulfonate.

Expression of ABAR and Truncated ABAR inEscherichia coli

The cDNAs encoding the full-length ABAR and three ABAR fragments

were amplified by PCR with the following primers: the forward primer

5#-GGAATTCTATGGCTTCGCTTGTGTATTCTC-3# and reverse primer

5#-ACGCGTCGACTTATCGATCGATCCCTTCGATCTTG-3# for full-length

ABAR; the forward primer 5#-GGAATTCTATGGCTTCGCTTGTGTATTCTC-3#and reverse primer 5#-ACGCGTCGACTTAATCAAGATTACATTGCTTAGC-3#for N772; the forward primer 5#-GGAATTCTCCCATGAGGCTGCTTTTC-

TCC-3# and reverse primer 5#-ACGCGTCGACTTATCGATCGATCCCTTCGA-

TCTTG-3# for C751; and the forward primer 5#-GGAATTCTCCCATGAGG-

CTGCTTTTCTCC-3# and reverse primer 5#-ACGCGTCGACTTATGTTGTGG-

GAATAGCCTGAGG-3# for C370 (without the sequence encoding the chloro-

plast transit peptide). The forward primers introduced an EcoRI restriction site,

and the reverse primers introduced a SalI restriction site into the fragments. The

PCR products were then digested and cloned directly into pET48b between the

EcoRI and SalI sites. The fragments in the plasmids were sequenced to check for

errors. The recombinant ABAR and ABAR fragments were expressed in E. coli

Rosetta gami2 (DE3; Novagen) strains as a 63His-ABAR (or truncated ABAR)

fusion protein. The E. coli strains containing the expression plasmids were

grown at 37�C in 1 L of Luria-Bertani medium containing 20 mg mL21

kanamycin until the optical density at 600 nm of the cultures was 0.6 to 0.8.

Protein expression was induced by the addition of isopropyl b-D-thiogalacto-

pyranoside to a final concentration of 1 mM at 16�C and 150 rpm. After 16 h, the

cells were lysed and proteins were purified on a Ni2+-chelating column as

described in the pET system manual.

Anti-Full-Length ABAR Serum Productionand Immunobloting

A standard immunization protocol was used to immunize female rabbits.

The E. coli-expressed, purified, 63His full-length ABAR fusion protein (2 mg)

was injected five times at intervals of 2 weeks. The antiserum was affinity

purified. The immunoblotting of the ABAR and truncated ABAR proteins

with the anti-ABAR serum was done essentially according to previously

described procedures (Shen et al., 2006). Proteins were separated by SDS-

PAGE on 10% polyacrylamide gels, and the polypeptides were transferred to

nitrocellulose membranes (0.45 mm; Amersham Life Science) in a medium

consisting of 25 mM Tris-HCl (pH 8.3), 192 mM Gly, and 20% (v/v) methanol.

After rinsing in Tris-buffered saline (TBS) containing 10 mM Tris-HCl (pH 7.5)

and 150 mM NaCl, the blotted membranes were preincubated for 3 h in a

blocking buffer containing 3% (w/v) bovine serum albumin dissolved in TBS

supplemented by 0.05% (v/v) Tween 20 (TBST1) and then incubated with

gentle shaking for 2 h at room temperature in appropriate antibodies (diluted

1:2,000 in the blocking buffer). Following extensive washes by TBST1, the

membranes were incubated with goat anti-rabbit IgG conjugated with alkaline

phosphatase (diluted 1:500 in TBST1) at room temperature for 1 h and then

washed with TBST2 (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% [v/v]

Tween 20) and TBS. The locations of antigenic proteins were visualized by

incubating the membranes with nitroblue tetrazolium and 5-bromo-4-chloro-

3-indolyl phosphate.

To test the specificity of the purified anti-ABAR serum, we preincubated

the antiserum (diluted 1:2,000 in the blocking buffer as described above) with

0.5% (w/v) purified ABAR fusion protein at 0�C for 30 min before use to

incubate the protein sample as described, and as a control, 5% (w/v) bovine

serum albumin was used in the same preincubation instead of the ABAR

protein. This preincubation of the purified anti-ABAR serum with the antigen

ABAR protein completely abolished the ability of the antiserum to recognize

either E. coli-expressed, purified, ABAR protein or natural ABAR protein from

Arabidopsis total protein, but the preincubation of the antiserum with bovine

serum albumin did not affect this antiserum-antigen recognition (Supple-

mental Fig. S4). This test showed that the anti-ABAR serum is specific to

ABAR protein.

Wu et al.




Preparation of the Arabidopsis Total Protein

The leaves of Arabidopsis were harvested from 3-week-old plants and

ground in liquid nitrogen. The sample was then transferred into an Eppendorf

tube containing ice-cold extraction buffer (1 mL g21 sample) consisting of, for

ABA-affinity chromatography, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM

EDTA, 0.1% Triton X-100, 10% glycerol, 2 mM 1,4-dithiothreitol, 1 mM phenyl-

methylsulfonyl fluoride, 5 mg mL21 leupeptin, 5 mg mL21 pepstatin A, and 5

mg mL21 aprotinin. The buffer for immunoblotting was composed of 50 mM

Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 10% glycerol,

and 13 protease inhibitor cocktail (Roche). The sample was extracted for 3 h in

ice. The extracts were centrifuged for 20 min at 16,000g, the supernatant was

transferred to a new Eppendorf tube and centrifuged again at 16,000g for 20

min, and then the concentration of the supernatant was detected by Coomas-

sie Brilliant Blue G-250 (Amresco). The samples were either kept at 0�C for

immediate use or frozen and stored at 280�C until use.

ABA Binding Assay with Affinity Chromatography

ABA was linked to EAH-Sepharose 4B via C1 (-COOH) of the ABA

molecule according to previously described procedures (Zhang et al., 2002).

(6)ABA (1 g; Sigma) dissolved in 60 mL of 50% (w/v) dimethylformamide

(Sigma) solution was mixed with 50 mL of drained EAH-Sepharose 4B

(GE Healthcare). 1-Ethyle-3-(3-dimethylaminopropyl)-carbodiimide hydro-

chloride (4 g; Sigma) was added into the ABA-EAH-Sepharose 4B solution,

the pH of which was adjusted to 8.0 with 1 N NaOH. The ABA-EAH-

Sepharose 4B solution was shaken for 20 h at 4�C in the dark. The gel was then

washed with 50% (w/v) dimethylformamide and then with both washing

buffer I consisting of 0.5 M NaCl and 0.1 M Tris-HCl (pH 8.3) and washing

buffer II composed of 0.5 M NaCl and 0.1 M sodium acetate-acetic acid (pH 4.0).

The gel was washed again with double distilled water. The coupling amount

of ABA to EAH-Sepharose 4B is determined essentially according to Nilsson

and Mosbach (1984). ABA-EAH-Sepharose 4B (40 mg) was dissolved in 80%

(v/v) glycerol, and then the UVA252 of the solution was measured with a UV-

Photometer (UV-240; Shimadzu) using 80% (v/v) glycerol as the control. The

amount of the coupled ABA was calculated according to the standard UV

absorbance per millimolar ABA at 252 nm. The tested coupling efficiency,

which was the ratio of the coupling amount of ABA to the total amount of the

amino groups conjugated to the gel, was approximately 60% to 70%.

All of the procedures described below were done at 4�C. The ABA-linked

EAH Sepharose 4B gel (1 mL) was first equilibrated with buffer A solution

consisting of 10 mM MES-NaOH (pH 6.5), 150 mM NaCl, 2 mM MgCl2, 2 mM

CaCl2, and 5 mM KCl. The equilibrated ABA-linked Sepharose 4B was then

incubated with 0.5 mL of pure E. coli-expressed ABAR protein (2 mg mL21) or

2 mL of Arabidopsis total protein (1 mg mL21) for 60 min in 5 mL of buffer A.

The final volume was 6.5 mL. For the competition assays of ABAR binding to

the ABA-affinity column, (+)ABA (Sigma) at 50 or 500 nM concentration was

added into buffer A for this incubation of the ABA-linked Sepharose 4B gel

with ABAR protein. The same amount of ethanol for solubilizing 50 or 500 nM

(+)ABAwas used instead of (+)ABA as a control. The ABA-linked Sepharose

4B gel mixed with ABAR protein (or with ABA in the competition assays) was

then packed into a column of 1.6 3 10 cm for affinity chromatography. The

column was first eluted with 150 mM NaCl in buffer A (36 mL, divided into 12

times with 3 mL each) to remove the unspecific bound proteins. ABA-binding

proteins were then eluted with the same buffer A containing 100 or 150 mM

NaCl and 4 mM (6)ABA (or the same concentration of other potential

agonists/antagonists as indicated). The eluting solutions were assayed for

immunoblotting with the anti-ABAR serum.

3H-Labeled ABA in-Buffer Binding Assay

3H-labeled ABA binding was performed essentially as described previ-

ously (Zhang et al., 2002; Shen et al., 2006) with modifications. We replaced the

Dextran T70-coated charcoal with a glass fiber filter to remove free [3H]ABA

from the binding medium. We previously used the ABA-binding technique

with a filter (Zhang et al., 1999, 2001), and this technique was recently used to

assay the ABA receptors GTG1 and GTG2 (Pandey et al., 2009).

[3H](+)ABA was made by American Radiolabeled Chemicals (2.37 3 1012

Bq mmol21; purity, 98.4%). The binding medium was composed of 50 mM

MES-NaOH (pH 7.0), 2 mM MgCl2, 1 mM CaCl2, 1 mM 1,4-dithiothreitol, 250

mM mannitol, and 10 mg mL21 protease inhibitor cocktail (Sigma). Each

binding assay contained 10, 20, 40, or 60 nM [3H]ABAwith or without a 1,000-

fold molar excess of unlabeled (6)ABA (Sigma), 2 mg of purified E. coli-

expressed ABAR/CHLH (or truncated ABAR/CHLH protein as indicated),

and binding buffer in a 200-mL total volume. The mixtures were incubated at

25�C for 60 min. The bound and free [3H]ABAwere separated, as mentioned

above, by filtering the mixture through a GF/F glass fiber filter (Whatman)

and washing with 3 mL of ice-cold ABA-binding buffer. The [3H]ABA bound

to ABAR protein retained by the filter was then quantified by scintillation

counting. The specific binding was determined by subtracting the binding in

the presence of a 1,000-fold molar excess of unlabeled (6)ABA (unspecific

binding) from total binding [the binding in the absence of unlabeled (6)ABA].

It is noteworthy that the radioactivity retained by the glass fiber filter (the

control in the absence of ABAR protein) should be subtracted from the total

and unspecific binding. The unspecific binding in all of the assays was less

than 10% of the total binding. The ABA-binding activity was expressed as

moles of [3H]ABA per mole of protein.

Transient Expression in Arabidopsis Protoplasts

For observation of the subcellular localization of the truncated ABAR

(ABARn, C751, the transit peptide-linked C370, and N772), the corresponding

cDNA fragments, driven by the cauliflower mosaic virus 35S promoter and

downstream tagged by GFP, were obtained by enzymatic degradation from

the above-mentioned binary vector pCAMBIA1300-221, which was created for

generating transgenic plants and harbors these cDNA fragments linked to

the 35S promoter in the 5# end and a C-terminal GFP flag in the 3# end. Each of

the 35S promoter-driven and GFP-tagged cDNA fragments was fused to the

pMD-19-T vector (Takara, Dalian Division) with the SphI (5# end) and EcoRI

(3# end) sites. Protoplasts were isolated from the leaves of 3- to 4-week old

plants of Arabidopsis (Col-0) and transiently transformed using polyethylene

glycol essentially according to an established protocol (http://genetics.mgh.

harvard.edu/sheenweb/). Fluorescence of GFP was observed with a confocal

laser scanning microscope (Zeiss; LSM 510 META) after incubation at 23�Cfor 16 h.

Phenotypic Analysis

Phenotypic analysis was done essentially as described previously (Shen

et al., 2006). For the germination assay, approximately 100 seeds each from the

wild type (Col-0), mutants, or transgenic mutants were sterilized and planted

in triplicate on MSmedium (Sigma; product no. M5524; full-strengthMS). The

medium contained 3% Suc and 0.8% agar (pH 5.9) and was supplemented

with or without different concentrations of (6)ABA. The seeds were incubated

at 4�C for 3 d before being placed at 20�C under light conditions, and

germination (emergence of radicles) was scored at the indicated times.

For the seedling growth experiment, seeds were germinated after strati-

fication on common MS medium and transferred to MS medium supple-

mented with different concentrations of (6)ABA in the vertical position. The

time for transfer was about 45 to 48 h after stratification for the transgenic

plants and 48 to 50 h for the assays of the abar-2, abar-3, and cch mutants. This

narrow ABA-responsive window of around 48 h in seedling growth was

observed for all of the ABAR-related mutants (including transgenic lines),

which may be partly associated with ABI5 regulation (Shen et al., 2006).

Seedling growth was investigated at the indicated times after the transfer, and

the lengths of primary roots were measured using a ruler. Seedling growth

was also assessed by directly planting the seeds in ABA-containingMSmedium

to investigate the response of seedling growth to ABA after germination.

For stomatal aperture assays, 3-week-old leaves were used. To observe

ABA-induced stomatal closure, leaves were floated in the buffer containing 50

mM KCl and 10 mM MES-Tris (pH 6.15) under a halogen cold light source

(Colo-Parmer) at 200 mmol m22 s21 for 2.5 h followed by the addition of

different concentrations of (6)ABA. Apertures were recorded on epidermal

strips after 2.5 h of further incubation to estimate ABA-induced closure. To

study ABA-inhibited stomatal opening, leaves were floated on the same buffer

in the dark for 2.5 h before they were transferred to the cold light for 2.5 h in

the presence of ABA, and then apertures were determined.

Chlorophyll and Porphyrin Measurements

The contents of chlorophyll, ProtoIX, and Mg-ProtoIX were assayed

essentially according to previously described procedures (Mochizuki et al.,

2001).





Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Transformation of wild-type and cch mutant

plants with empty vector (harboring GFP) did not alter ABA sensitivity

in seed germination, seedling growth, and stomatal aperture.

Supplemental Figure S2. Affinity chromatography shows that both barley

XanF and rice CHLH bind ABA.

Supplemental Figure S3. Alignment of deduced amino acid sequences of

CHLH (Os03g20700) and a CHLH-like protein (Os07g46310) in rice.

Supplemental Figure S4. Purification of E. coli-expressed ABAR/CHLH

protein and test of the specificity of the anti-ABAR/CHLH serum.

Supplemental Table S1. Raw data (cpm) for ABA-binding parameters

presented in Figure 1, J and K.

Supplemental Materials and Methods S1.

ACKNOWLEDGMENT

We thank Dr. M. Hansson (Carlsberg Laboratory, Copenhagen, Denmark)

for kindly providing the plasmid pET15bXanF.

Received May 1, 2009; accepted June 14, 2009; published June 17, 2009.

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