gg1 gg2 gb: heterotrimeric g protein g -deﬁcient mutants ... · gg1 1 gg2 gb: heterotrimeric g...

Gg1 1 Gg2 � Gb: Heterotrimeric G Protein Gg-DeficientMutants Do Not Recapitulate All Phenotypes ofGb-Deficient Mutants1[C][W][OA]

Yuri Trusov, Wei Zhang, Sarah M. Assmann, and Jose Ramon Botella*

Plant Genetic Engineering Laboratory, Department of Botany, School of Integrative Biology, University ofQueensland, Brisbane, Queensland 4072, Australia (Y.T., J.R.B.); and Biology Department, PennsylvaniaState University, University Park, Pennsylvania 16802–5301 (W.Z., S.M.A.)

Heterotrimeric G proteins are signaling molecules ubiquitous among all eukaryotes. The Arabidopsis (Arabidopsis thaliana)genome contains one Ga (GPA1), one Gb (AGB1), and two Gg subunit (AGG1 and AGG2) genes. The Gb requirement of afunctional Gg subunit for active signaling predicts that a mutant lacking both AGG1 and AGG2 proteins should phenotyp-ically resemble mutants lacking AGB1 in all respects. We previously reported that Gb- and Gg-deficient mutants coincideduring plant pathogen interaction, lateral root development, gravitropic response, and some aspects of seed germination. Here,we report a number of phenotypic discrepancies between Gb- and Gg-deficient mutants, including the double mutant lackingboth Gg subunits. While Gb-deficient mutants are hypersensitive to abscisic acid inhibition of seed germination and arehyposensitive to abscisic acid inhibition of stomatal opening and guard cell inward K1 currents, none of the available Gg-deficient mutants shows any deviation from the wild type in these responses, nor do they show the hypocotyl elongation andhook development defects that are characteristic of Gb-deficient mutants. In addition, striking discrepancies were observed inthe aerial organs of Gb- versus Gg-deficient mutants. In fact, none of the distinctive traits observed in Gb-deficient mutants(such as reduced size of cotyledons, leaves, flowers, and siliques) is present in any of the Gg single and double mutants.Despite the considerable amount of phenotypic overlap between Gb- and Gg-deficient mutants, confirming the tightrelationship between Gb and Gg subunits in plants, considering the significant differences reported here, we hypothesize theexistence of new and as yet unknown elements in the heterotrimeric G protein signaling complex.

Heterotrimeric G proteins contain Ga, Gb, and Ggsubunits and transduce signals from activated plasmamembrane receptors to intracellular effectors (Gilman,1987). Upon activation of the receptor, GDP bound toinactive Ga is exchanged for GTP, causing a confor-mational change that leads to activation with or with-out physical dissociation of the Ga subunit from theGbg complex (Rebois et al., 1997; Klein et al., 2000;Bunemann et al., 2003; Adjobo-Hermans et al., 2006;Digby et al., 2006). The activated subunits then trans-mit the signal to their specific effector molecules untilintrinsic GTPase activity of the Ga subunit hydrolyzesthe GTP molecule, thus returning Ga to its inac-tive state and sequestering Gbg back to the inactive

heterotrimer. Gb and Gg subunits form tightly bounddimers that work as functional units and can only bedissociated under strong denaturing conditions(Schmidt et al., 1992; Clapham and Neer, 1993; Gautamet al., 1998; McCudden et al., 2005).

In animal systems, G proteins mediate the signalingof over 800 receptors (G protein-coupled receptors;Pierce et al., 2002; Fredriksson et al., 2003; Zhang et al.,2006). Multiple family members exist for each of thethree subunits, and different combinatorial possibili-ties provide the required specificity for multitudi-nous G protein-based signaling pathways (Gautamet al., 1998; Balcueva et al., 2000; Wettschureck andOffermanns, 2005; Marrari et al., 2007). In open con-trast, plants only contain one or two genes for each ofthe subunits (Ma et al., 1990; Poulsen et al., 1994; Weisset al., 1994; Gotor et al., 1996; Iwasaki et al., 1997; Seoet al., 1997; Marsh and Kaufmann, 1999; Ando et al.,2000; Mason and Botella, 2000, 2001; Perroud et al.,2000; Kang et al., 2002; Hossain et al., 2003a, 2003b; Katoet al., 2004; Misra et al., 2007). In Arabidopsis (Arabi-dopsis thaliana), a single Ga (Ma et al., 1990; Ma, 1994), asingle Gb (Weiss et al., 1994), and two Gg (Mason andBotella, 2000, 2001) subunit genes have been identified.

Despite the fact that only two combinatorial possi-bilities are feasible for the heterotrimers in Arabidop-sis, G proteins are involved in multiple processes(Assmann, 2004; Jones and Assmann, 2004; Perfus-Barbeoch et al., 2004). Recent genetic studies using

1 This work was supported by the Australian Research Council(Discovery Grant nos. DP0344924 and DP0772145), the U.S. Depart-ment of Agriculture (grant no. 2006–35100–17254), and the NationalScience Foundation (grant no. MCB–0209694).

* 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:Jose Ramon Botella ([email protected]).

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

[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.108.117655

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Ga- and Gb-deficient or -overproducing mutants havedemonstrated the involvement of G proteins in ab-scisic acid (ABA) and brassinosteroid sensitivity duringseed germination and early plant development (Ullahet al., 2002; Lapik and Kaufman, 2003; Chen et al., 2004,2006; Pandey et al., 2006; Warpeha et al., 2006), stoma-tal regulation (Wang et al., 2001), D-Glc signaling(Huang et al., 2006; Wang et al., 2006), light perception(Okamoto et al., 2001; Warpeha et al., 2006, 2007),rosette leaf, flower, and silique development (Leaseet al., 2001; Ullah et al., 2003), plant defense againstnecrotrophic fungi (Llorente et al., 2005; Trusov et al.,2006), and auxin signaling in roots (Ullah et al., 2003;Trusov et al., 2007). Similar functional multiplicity hasbeen observed in rice (Oryza sativa; Ashikari et al.,1999; Fujisawa et al., 1999; Ueguchi-Tanaka et al., 2000;Suharsono et al., 2002; Komatsu et al., 2004; Oki et al.,2005).

In animals and fungi, the existence of a functionalGg subunit is a compulsory prerequisite for the func-tioning of the entire heterotrimer, and lack of Ggsubunits results in the obliteration of both Gbg- andGa-mediated pathways (Gilman, 1987; Kisselev et al.,1994; Manahan et al., 2000; Krystofova and Borkovich,2005; Myung et al., 2006). There is one notable excep-tion to this rule: the human neurospecific Gb5 subunitforms functional dimers with some members of theregulator of G protein signaling (RGS) subfamily Cproteins instead of with conventional Gg subunits(Snow et al., 1998; Sondek and Siderovski, 2001). PlantG proteins appear to behave similarly to their animalcounterparts, and tight physical interaction betweenGb and each of the Gg subunits has been demon-strated in vitro (Mason and Botella, 2000, 2001) and invivo (Kato et al., 2004; Adjobo-Hermans et al., 2006).Moreover, it has been shown that Arabidopsis Gbsubunit localization on the plasma membrane requiresa Gg subunit (Obrdlik et al., 2000; Adjobo-Hermanset al., 2006). Evidence for functional interaction of thesubunits was recently provided using overexpressionof a truncated Gg1 subunit lacking the isoprenylationmotif (which is responsible for anchoring the bg dimerto the membrane) and mutants lacking each of or bothGg subunits (Chakravorty and Botella, 2007; Trusovet al., 2007). It has also been shown that different Ggsubunits confer specificity to the Gbg dimer, with theGbg1 dimer mediating signal transduction eventsduring plant defense against necrotrophic fungi,acropetal auxin signaling in roots, and osmotic stressregulation of seed germination, while Gbg2 is in-volved in basipetal auxin signaling in roots and D-Glcsensing during germination (Trusov et al., 2007).

However, despite the above-mentioned similaritiesbetween plant and animal G proteins, a number ofunusual properties observed in the plant subunitshave led to suggestions that, in some cases, the animalparadigm may not necessarily hold true in plants. Forinstance, it was established that unlike animal sub-units, Arabidopsis Ga and Gbg are capable of tether-ing to the plasma membrane independently and do

not rely on each other (Adjobo-Hermans et al., 2006;Zeng et al., 2007; Wang et al., 2008). It was also shownthat the GTPase activity of the Arabidopsis Ga subunitis very low, leading to the hypothesis by some authorsthat Ga might be in the activated state by default(Willard and Siderovski, 2004; Johnston et al., 2007a;Temple and Jones, 2007). In addition, the interactionbetween Ga and the Gbg dimer in rice has beenreported to be relatively weak compared with that inanimal systems (Kato et al., 2004), although formationof the heterotrimer in vivo has been demonstrated(Kato et al., 2004; Adjobo-Hermans et al., 2006). Re-cently, it was shown that in Arabidopsis both Ga andGb are associated with large macromolecular com-plexes of approximately 700 kD (Wang et al., 2008).Finally, Arabidopsis Gg subunits are capable of beingtargeted to the plasma membrane in mutants lackingfunctional Ga and Gb subunits (Zeng et al., 2007).

Here, we present data conflicting with the estab-lished heterotrimeric G protein model. We found that anumber of phenotypic alterations observed in Ga- orGb-deficient mutants cannot be detected in mutantslacking Gg1, Gg2, or both Gg subunits. Our resultsraise the possibility that Gg subunits are not requiredfor some Ga- and Gb-mediated processes in Arabi-dopsis or that additional nonconventional Gg subunitsexist in this species.

RESULTS

The Expression Profiles of AGG1 and AGG2 in

Reproductive Organs Do Not Match the AGB1Expression Pattern

We previously reported that GUS staining patternsin transgenic Arabidopsis ecotype Columbia (Col-0)plants carrying promoter fusions of each of the AGG1and AGG2 genes with the GUS reporter gene weretissue specific and that together they overlap AGB1expression patterns in most plant tissues and devel-opmental stages (Anderson and Botella, 2007; Trusovet al., 2007). Nevertheless, a number of small butimportant differences can also be observed, especiallyin reproductive tissues. Analysis of GUS expression inflowers and siliques revealed that AGB1 is moderatelyexpressed in sepals and stamen filaments (Fig. 1A),with high expression found in stigma and pollen(Fig. 1, B and C). In siliques, GUS staining was ob-served at both ends, gradually disappearing towardthe center (Fig. 1D). In AGG1:GUS plants, GUS ex-pression was only detected in the stigma of matureflowers (Fig. 1, B and C), possibly correlating withpollination, while in siliques only the abscission zoneshowed staining (Fig. 1D). In AGG2:GUS transform-ants, GUS expression was evident in the apex ofstamen filaments at a very early developmental stageand disappeared before the flower opened (Fig.1C). No GUS staining was detected in siliques ofAGG2:GUS plants. Discrepancies were also observedin germinating seeds, where distinct GUS staining in

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AGB1:GUS plants was observed much earlier (24 and48 h after imbibition) than in AGG1:GUS or AGG2:GUSplants. However, expression in AGG1:GUS or AGG2:GUS increased to detectable levels and overlappedwith AGB1 expression in 4-d-old seedlings (Fig. 1E), inagreement with a previous report (Trusov et al., 2007).

ABA Sensitivity of Gg1- and Gg2-Deficient Mutants

in Germination

Heterotrimeric G protein involvement in differentaspects of seed germination has been established by anumber of studies (Ullah et al., 2002; Lapik andKaufman, 2003; Chen et al., 2004, 2006; Pandey et al.,2006; Liu et al., 2007; Warpeha et al., 2007). Involvementof the heterotrimeric G protein signaling componentsGPA1, AGB1, GCR1, and RGS1 in ABA inhibition ofseedgermination is well documented (Ullah et al., 2002;

Chen et al., 2004, 2006; Pandey et al., 2006). It wasrecently proposed that a second putative G protein-coupled receptor (GCR2) is a plasma membrane recep-tor for ABA (Liu et al., 2007); however, these resultshave been challenged (Gao et al., 2007; Johnston et al.,2007b; Illingworth et al., 2008).

Roles for the Arabidopsis Gg subunits, AGG1 andAGG2, in D-Glc and osmotic sensing during germina-tion were recently reported (Trusov et al., 2007), but nodata are available at present on their involvement inABA sensing. Therefore, we compared the responsesof Ga-, Gb-, and Gg-deficient mutants to ABA-mediatedinhibition of germination (Fig. 2). We analyzed germi-nation rates for seven different seed lots for eachgenotype (stored in an identical environment for ap-proximately 1 month after harvest) under a number ofexperimental conditions. Each lot was tested at leasttwice. In all tests, all genotypes showed 100% germi-nation on control medium (no ABA added) by day 3after light exposure (data not shown). In accordancewith previous reports showing hypersensitivity ofGa- and Gb-deficient mutants to ABA during germina-tion (Pandey et al., 2006), gpa1-4 and agb1-2 mutantsshowed enhanced ABA-mediated inhibition of germi-nation compared with the wild type (Fig. 2, A and B).In contrast, four independent single Gg-deficient mu-tants, agg1-1c, agg1-2, agg2-1, and agg2-2, as well as thedouble agg1 agg2 mutant showed levels of ABA sen-sitivity similar to wild-type plants and in one case(agg1-2 in 5 mM ABA) even showed hyposensitivity(P , 0.05; Fig. 2, A and B). In some isolated experiments,Gg1- or Gg2-deficient mutants showed either de-creased or slightly increased ABA sensitivity com-pared with the wild type, but the responses neverreached the hypersensitivity levels displayed by Gb-deficient mutants. Figure 2, A and B, shows germina-tion rates for the wild type and all mutants in arepresentative experiment. The differences in ABAsensitivity between gpa1-4 and agb1-2 mutants and allof the other genotypes analyzed (the wild type andGg-deficient mutants) were statistically significant(P , 0.05).

It is known that high sugar concentration inhib-its germination, while low amounts of Suc or Glccan rescue ABA-mediated inhibition of germination(Garciarrubio et al., 1997; Price et al., 2003). We ana-lyzed germination rates on ABA-containing mediumin the presence or absence of 2% Suc. Suc increased thegermination of all genotypes at the two ABA concen-trations assayed (Fig. 2, A and B), although, due to thecomplexity of the figure, it is difficult to visualize andcompare the effects of Suc in the different genotypes.One useful way to visualize differences in behavior isto plot the relative effect of Suc on germination inhi-bition by ABA (Fig. 2C). Of the two ABA concentra-tions studied, only one (5 mM ABA) is amenable to thistype of analysis, since in these specific experimentalconditions, the percentage of germinated seedsshowed a linear increase during the studied period(3–6 d after induction). In contrast, using 2 mM ABA,

Figure 1. AGB1, AGG1, and AGG2 expression patterns in flowers.Histochemical analysis of GUS expression in transgenic Arabidopsisplants carrying AGB1:GUS, AGG1:GUS, or AGG2:GUS fusions. A,Fully opened flowers. B, Stigma of young (unopened bud) and matureflowers. C, Anther, stamen filaments, and pollen. D, Green-stagesiliques. E, Germinating seedlings at 24, 48, and 96 h after imbibition.

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638 Plant Physiol. Vol. 147, 2008



almost 100% germination was observed on days 5 to 6for most genotypes. This allowed us to calculate the‘‘rescue’’ effect of Suc as the relative increase in ger-mination [e 5 (s 2 n)/s, where s is the percentage ofgerminated seeds on Suc-containing plates and n is thepercentage of germinated seeds on plates without Suc]and average it for the four time points. Both gpa1-4and agb1-2 mutants showed statistically significantlyhigher values than the remaining genotypes (P , 0.05),although absolute germination levels remained signif-icantly (P , 0.05) lower for these genotypes than for allothers.

Gg-Deficient Mutants Do Not Display the agb1-2Deetiolated Phenotype in Darkness

Partially deetiolated seedlings of Ga- and Gb-deficientmutants have short hypocotyls with visibly increasedgirth and a characteristic open hook (Ullah et al., 2001,2003; Wang et al., 2006). We analyzed hypocotyl elon-gation rate and hook development in darkness as wellas light inhibition of hypocotyl elongation in gpa1-4,agb1-2, agg1-1c, agg1-2, agg2-1, agg2-2, and agg1 agg2

mutants as well as in the wild type. To ensure syn-chronized germination, all seeds were stratified for 5 dand induced to germinate under 150 mmol m22 s21

continuous light during 24 h. Afterward, plates withseeds were placed vertically either in a dark cabinet orunder continuous light (90 mmol m22 s21) for 24, 48,and 72 h.

Figure 3, A and B, shows hypocotyl elongationdynamics of all mutant genotypes and the wild typegrown in darkness and light, respectively. In agree-ment with previous reports (Ullah et al., 2001, 2003),hypocotyl elongation rates for Ga- and Gb-deficientmutants (gpa1-4 and agb1-2) in darkness were lowercompared with those for the wild type, with statisti-cally significant differences after 24 and 48 h (P , 0.01and P , 0.05, respectively). Under light, gpa1-4 andagb1-2 hypocotyls were significantly shorter than wild-type hypocotyls at all three time points (at least P ,0.05). In open contrast to the behavior of gpa1-4 andagb1-2 mutants, hypocotyl elongation in either dark orlight conditions in each of the individual Gg1- or Gg2-deficient mutants or the double agg1 agg2 mutant wasnot statistically different from that in the wild type.

Figure 2. Sensitivity to ABA-in-duced inhibition of seed germina-tion in G protein complex mutants.A and B, Seeds from matched seedlots were surface sterilized andplated on 0.53 Murashige andSkoog medium plates in the pres-ence of 2 mM (A) or 5 mM (B) ABA.Plates were transferred to 100 mmolm22 s21 light and 23�C. Germina-tion was recorded at 3, 4, 5, and 6 dafter transfer of the plates under lightand expressed as a percentage oftotal number of seeds. The experi-ment was repeated three times, anddata were averaged (n . 100 foreach experiment). The error barsrepresent SE. C, Rescue effect ofSuc on germination inhibited by5 mM ABA. Error bars indicate SE

values obtained from four measure-ments. Asterisks indicate values sta-tistically significantly different fromthe wild type (P , 0.05).





When the hook angle was measured after 24 h ofdark incubation, gpa1-4 and agb1-2 mutants showedthe typical ‘‘open-hook’’ phenotype described previ-ously (Ullah et al., 2003; Fig. 2C). In contrast, all of thesingle Gg-deficient mutants as well as the double agg1agg2 mutant showed a wild-type phenotype clearlydifferent from those of gpa1-4 and agb1-2.

None of the Morphological Alterations of Aerial OrgansObserved in Ga- and Gb-Deficient Mutants Is Present in

Gg-Deficient Mutants

Phenotypic analyses of Ga- and Gb-deficient mu-tants have revealed a number of developmental andmorphological abnormalities (Ullah et al., 2003). Inorder to determine whether Gg-deficient mutants

showed similar traits, we compared the wild type,gpa1-4, agb1-2, agg1-1c, agg1-2, agg2-1, agg-2-2, and agg1agg2 grown under ‘‘standard’’ conditions as specifiedin ‘‘Materials and Methods.’’ Quantitative analysis of anumber of morphological characteristics was carriedout at defined growth stages (Boyes et al., 2001) duringdevelopment (Table I; Fig. 4). Mean values of thesetraits in the mutant lines were analyzed for significantdeviation from the corresponding values in the wildtype by pair-wise, two-sample Student’s t test. Unlessstated otherwise, the mean values were derived fromanalysis of at least 30 plants grown in a checkerboardpattern.

Differences between agb1-2 and wild-type plantsbecome apparent from a very early stage of develop-ment. Smaller and rounder cotyledons of agb1-2 mu-

Figure 3. Seedling development of G protein com-plex mutants grown in darkness or under light. A andB, Hypocotyl elongation rates of the wild type and themutants at 24, 48, and 72 h after germination indarkness (A) or under 90 mmol m22 s21 light (B). Errorbars indicate SE. At least 20 seedlings were measured.C, Degree of hook opening in dark-grown wild-typeand mutant seedlings measured approximately 24 hafter germination. SE values indicated by error bars arebased on a minimum of 20 seedlings. Closed hookswere treated as having zero degree of opening. Tripleasterisks indicate values statistically significantly dif-ferent from the wild type (P , 0.001). The inset showsrepresentative seedlings in order from left to right:Col-0, gpa1-4, agb1-2, agg1-1c, agg1-2, agg2-1,agg2-2, and agg1 agg2.

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tants are apparent as early as 7 d after germination(Fig. 4A). The shape of the gpa1-4 cotyledons is verysimilar to that of wild-type cotyledons at this stage,although they are larger (Fig. 4A). In contrast, all of thesingle agg1-1c, agg1-2, agg2-1, and agg2-2 mutants aswell as the double agg1 agg2 mutant are indistinguish-able from the wild type at this developmental stage(Fig. 4A). Measurements of at least 50 seedlings of eachgenotype revealed that these visible differences be-tween agb1-2 or gpa1-4 and the wild type are statisti-cally significant (P , 0.001), while all Gg-deficientmutants were indeed similar to the wild type (Table I).Rosette diameter measured at the inflorescence emer-gence stage was smaller in agb1-2 mutants (P , 0.001),partly due to the shorter size of the petioles (P , 0.001;Table I). The shorter petiole can be observed very earlyin Gb-deficient mutants, giving a distinctive appear-ance caused by the cotyledons being very close to thehypocotyl (Fig. 4A). The rosette leaves of gpa1-4 andagb1-2 mutants have a characteristic crinkled surfaceand rounder appearance compared with those of thewild type (Fig. 4B). The ratios between leaf length andwidth in Ga- and Gb-deficient mutants were signifi-cantly lower than in the wild type (P , 0.001; Table I).In contrast, neither the individual Gg-deficient mu-tants nor the double agg1 agg2 mutant showed anystatistically significant differences from the wild typefor any of the above-mentioned traits: rosette diameter,leaf appearance, petiole size, and leaf length-widthratio.

Inflorescence emergence, defined by the appearanceof flower buds, occurs approximately 2 d earlier inagg1-1c, agg1-2, and double agg1 agg2 mutants thanin the wild type and gpa1-4, agg2-1, and agg2-2 mu-tants. The agb1-2 mutants showed delayed flowering,with inflorescence emergence occurring 2 d later than

in the wild type (Table I). Despite the delay in inflo-rescence emergence (P , 0.05), the first agb1-2 floweropened at the same time as for the wild type, gpa1-4,and Gg2-deficient mutants (data not shown). The finalinflorescence height was noticeably lower in agb1-2 (P, 0.01), while all other mutants were similar in heightto the wild type. Apical dominance is known to beregulated by basipetal auxin flow from the apicalmeristem (Jones, 1998; Dun et al., 2006; Leyser, 2006).Attenuation of auxin signaling by Gb and both Ggsubunits has been established previously in roots(Ullah et al., 2003; Trusov et al., 2007). In the floralstem, our data showed increased apical dominance inagb1 mutants, as evidenced by a decreased number ofbranches, which is consistent with previous reports(Ullah et al., 2003). In contrast, all of the Gg-deficientmutants showed a wild-type floral stem branchingpattern. Contrary to a previous report (Ullah et al.,2003), we found that the number of open flowers at themidflowering stage was higher in agb1-2 plants (P , 0.01)compared with all other genotypes analyzed (Table I).Flowers were significantly smaller in agb1-2 and gpa1-4mutants, while all Gg-deficient mutants had flowers ofsimilar size compared with the wild type (Table I; Fig.4C). Even though we measured only the diameter ofthe fully open flower, sepal size was also affected inagb1-2 and gpa1-4 mutants, as can be seen in Figure 4C.

Alteration of the silique shape was first describedfor the agb1-1 mutant (Lease et al., 2001), and similarobservations were made for agb1-2 (Ullah et al., 2003).Our measurements of agb1-2 concur with previousreports showing shorter and wider siliques (P ,0.001), with the characteristic blunt (flat) tips (Table I;Fig. 4D). Similarly to agb1-2, the gpa1-4 mutants pro-duced shorter (although to a lesser extent) and widersiliques, with the characteristic blunt tip. Curiously,

Figure 4. Comparison of characteristic morphologicaltraits in the wild type and gpa1-4, agb1-2, and Gg

mutants. A, Seven-day-old soil-grown seedlings. B,Average rosette leaves from a 30-d-old plant. C, Fullyopen mature flowers. D, Fully expanded siliques. [Seeonline article for color version of this figure.]





these results differ from those reported by Ullah et al.(2003), who analyzed two different Ga-deficient mu-tants, gpa1-1 and gpa1-2, and found their siliques to beslightly longer than wild-type siliques and having awild-type tip shape. It is noteworthy that gpa1-1 andgpa1-2 mutants were obtained in the Wassilewskija(Ws) ecotype, while gpa1-4 (as well as another Ga-deficient mutant, gpa1-3) is in the Col-0 background.To analyze the effects of growth conditions or ecotypein silique development, we simultaneously grewgpa1-1, gpa1-2, gpa1-3, and gpa1-4 mutants and corre-sponding wild-type plants. Interestingly, siliques ofgpa1-1 and gpa1-2 were indeed slightly longer thanthose of the Ws wild type, with ‘‘normal’’ acute tips, asdescribed by Ullah et al. (2003), while both gpa1-3 andgpa1-4 siliques were shorter than those in the Col-0wild type, and both had blunt tips (Table II). Thisobservation is quite important, as it demonstrates thatsome effects of GPA1 deficiency are dependent on thegenetic background and are not necessarily universal,even within the same species. Siliques in all Gg-deficient mutants showed wild-type characteristics.

In contrast to all other aerial organs, in which theknockout of AGB1 results in a reduced size, the siliquepeduncle was longer in agb1-2 mutants (Table I), andthis trait was even more pronounced in gpa1-4 plants(P , 0.001). As has been the case with most of themorphological traits studied here, all Gg-deficientmutants showed wild-type peduncle length (Table I).It is worth mentioning that, even though Ga-deficientmutants showed ecotype-dependent behavior for si-lique length and tip shape, the peduncles of gpa1-1,gpa1-2, gpa1-3, and gpa1-4 were almost twice as long asthose of the relevant Ws or Col-0 wild-type controlplants (P , 0.001; Table II). Importantly, pedunclelength values presented here were obtained from thefirst two to three (lowest) siliques per plant, as pedun-cle length decreases gradually in size toward the top ofthe inflorescence. Nevertheless, the described trends

were conserved along the entire inflorescence (datanot shown). The number of seeds per silique wascalculated using three siliques per plant and averagedfor 10 plants per genotype. Siliques of agb1-2 and gpa1-4mutants contained fewer seeds than wild-type con-trols (P , 0.001 and P , 0.01, respectively), while noneof the Gg-deficient mutants showed statistically sig-nificant difference from the wild type. The shorterinflorescence and higher number of siliques present inagb1-2 mutant plants resulted in a highly statisticallysignificant difference in the density of siliques (num-ber of siliques per centimeter of inflorescence), whileGa- and all Gg-deficient mutants were similar to thewild type (Table I).

Guard Cells of Gg-Deficient Mutants Show Wild-Type

Responses to ABA

ABA is a well-studied phytohormone in guardcell signaling and is an important component ofmany stress responses in plants. In Arabidopsis, Ga-deficient mutants show alterations in a number ofguard cell responses, such as hyposensitivity to ABAinhibition of stomatal opening and reduced ABAresponsiveness of guard cell inward K1 channels(Wang et al., 2001; Coursol et al., 2003; Mishra et al.,2006). Our recent studies (L.M. Fan, unpublished data)show that Gb-deficient mutants show the same alter-ations in guard cell ABA responses as observed forGa-deficient mutants. Therefore, we assessed bothABA inhibition of light-induced stomatal openingand the ABA promotion of stomatal closure in Gg-deficient plants. In agg1-1c, agg2-1, and agg2-2 singlemutants, stomatal responses to 50 mM ABA were notstatistically different from those of the wild type. Awild-type ABA response was also observed in thedouble agg1 agg2 mutant (Fig. 5, A and B). To ensurethat a subtle alteration in ABA sensitivity was notoverlooked in these experiments, the assays were

Table I. Morphological characterization of wild-type and mutant plants with altered heterotrimeric G proteins

*, P , 0.05. **, P , 0.01. ***, P , 0.001.

Characteristic Stage Col-0 gpa1-4 agb1-2 agg1-1c agg1-2 agg2-1 agg2-2 agg1 agg2

Cotyledons (mm) 1.0 6.7 6 0.1 7.5 6 0.1*** 5.5 6 0.1*** 6.9 6 0.2 6.5 6 0.1 6.9 6 0.1 6.6 6 0.1 6.9 6 0.2

Rosette/leaf

Length-width ratio 3.90 1.8 6 0.07 1.3 6 0.05*** 1.2 6 0.04*** 1.9 6 0.07 1.8 6 0.06 1.8 6 0.08 1.8 6 0.09 1.7 6 0.12

Petiole length (mm) 3.90 9.6 6 0.4 10.8 6 0.3** 6.7 6 0.3*** 9.3 6 0.3 9.4 6 0.4 8.9 6 0.5 9.7 6 0.3 8.8 6 0.4

Crinkly surface 3.90 No Yes Yes No No No No No

Rosette diameter (mm) 3.90 41.3 6 1.1 43.0 6 1.3 29.2 6 1.2*** 44.5 6 1.0 41.4 6 1.2 43.6 6 1.0 43.9 6 1.3 40.8 6 1.8

Inflorescence/flowers

Buds are visible (day) 5.10 22.7 6 1.1 23.1 6 1.3 24.9 6 0.9* 18.3 6 1.5* 19.3 6 0.8* 22.4 6 1.1 21.9 6 1.3 19.8 6 0.7*

Length (cm) 6.90 33.6 6 0.6 30.1 6 1.6 26.6 6 1.3** 32.2 6 1.4 31.2 6 1.2 32.3 6 0.8 31.2 6 1.1 32.3 6 1.8

No. of branches 6.90 3.4 6 0.4 3.5 6 0.6 2.4 6 0.5* 3.1 6 0.5 3.2 6 0.5 3.5 6 0.6 3.4 6 0.5 3.1 6 0.5

No. of open flowers 6.50 3.2 6 0.2 3.4 6 0.3 6.4 6 1.6** 3.2 6 0.6 2.9 6 0.4 2.9 6 0.6 3.1 6 0.3 3.5 6 0.4

Flower diameter (mm) 6.50 4.0 6 0.3 3.5 6 0.1* 3.3 6 0.2* 4.1 6 0.1 4.2 6 0.1 4.4 6 0.2 4.3 6 0.2 4.0 6 0.1

Silique

Length (mm) 6.90 13.8 6 0.3 12.7 6 0.3* 10.6 6 0.2*** 13.9 6 0.4 13.7 6 0.4 14.1 6 0.5 13.8 6 0.3 13.2 6 0.3

Width (mm) 6.90 0.73 6 0.02 0.82 6 0.03** 0.93 6 0.03*** 0.72 6 0.02 0.70 6 0.02 0.72 6 0.02 0.71 6 0.02 0.72 6 0.02

Blunt tip 6.90 No Yes Yes No No No No No

Peduncle length (mm) 6.90 7.8 6 0.3 13.2 6 0.5*** 9.9 6 0.3* 7.7 6 0.3 8.2 6 0.4 8.1 6 0.3 7.9 6 0.3 8.1 6 0.3

No. of seeds 8.00 51.6 6 1.3 44.0 6 1.2** 39.6 6 0.9*** 55.3 6 1.0 51.0 6 1.1 50.0 6 2.5 53.3 6 1.5 49.3 6 1.8

No. of siliques (per cm) 9.70 0.88 6 0.02 0.93 6 0.03 1.13 6 0.02*** 0.95 6 0.02 0.81 6 0.02 0.81 6 0.01 0.93 6 0.02 0.94 6 0.04

Trusov et al.




repeated with a lower concentration of ABA (20 mM).As shown in Figure 5, C and D, stomatal apertureresponses of the Gg-deficient single and double mu-tants at this lower ABA concentration still remainedindistinguishable from those of wild-type plants.

Many intracellular events contribute to the finaloutcome of an alteration in stomatal aperture. Onewell-defined aspect of the ABA inhibition of stomatalopening is inhibition of the K1 channels that mediateK1 uptake. To assess a more restricted ABA signalingpathway in guard cells, we used the electrophysiolog-ical technique of patch clamping to evaluate the ABAresponsiveness of inward K1 currents. Ga- and Gb-deficient mutants lack ABA inhibition of inward K1

currents (Wang et al., 2001; Coursol et al., 2003; L.M.Fan, unpublished data). By contrast, as shown inFigure 6, ABA inhibited the inward K1 currents ofall of the Gg-deficient single and double mutants to thesame extent as was observed for wild-type guard cells.As has been reported previously (Wang et al., 2001;Becker et al., 2003), no ABA regulation of the outwardK1 channels that mediate K1 efflux during stomatalclosure was observed in wild-type Arabidopsis plants,and the same absence of an ABA effect was observedin Ga- and Gb-deficient mutants (Wang et al., 2001;Coursol et al., 2003; L.M. Fan, unpublished data) aswell as in all of the Gg-deficient mutants (Fig. 6).

DISCUSSION

In the ‘‘canonical’’ model of heterotrimeric G proteinsignal transduction, activity of the Gb subunit reliesheavily on its binding to the Gg subunit and subse-quent membrane localization (Casey, 1995; Marrariet al., 2007). In plants, as in animals and fungi, the Gbsubunit of the heterotrimeric G protein requires inter-action with a Gg subunit for plasma membrane local-ization (Adjobo-Hermans et al., 2006; Zeng et al.,2007). In fact, both canonical Arabidopsis Gg subunitswere shown to be prenylated and localized to theplasma membrane (Adjobo-Hermans et al., 2006; Zenget al., 2007). Also similar to animals, plant Gb subunitsare tightly bound to Gg subunits, as shown in vitro(Mason and Botella, 2000, 2001) and in vivo (Katoet al., 2004; Adjobo-Hermans et al., 2006). Moreover,overexpression of a mutated AGG1 lacking the iso-prenylation motif resulted in a phenotype that resem-bles that of Gb-deficient mutants (Chakravorty andBotella, 2007). These facts compel us to hypothesizethat in plants, as in animals, the bg subunits act as a

functional monomer. Therefore, Arabidopsis plantslacking either or both of the two known Gg subunits(AGG1 or AGG2) should display phenotypes thattotally or partially overlap those observed in mutantslacking AGB1. Furthermore, a double AGG1 AGG2knockout is expected to be identical to the AGB1-deficient mutants in all respects. Indeed, this is thecase in many instances, and we previously reportedthat lateral root formation, resistance to necrotrophicpathogens, and germination on 6% Glc are similarlyaltered in Gb- and Gg-deficient mutants (Trusov et al.,2007). Additionally, the expression patterns of AGG1and AGG2 resembled AGB1 expression in most planttissues (Trusov et al., 2007). On the other hand, theunique ability of the plant Gg subunits to localize tothe plasma membrane independently of Gb (Adjobo-Hermans et al., 2006; Zeng et al., 2007; Wang et al.,2008) raises the possibility that in Arabidopsis Gb-lacking mutants, free Gg subunits could be involved inabnormal interactions. However, in many cases, thisremote possibility could be ruled out by comparingGb-deficient mutants with Ga-deficient mutants pos-sessing Gbg dimers and not free Gg subunits.

Here, we show that there are considerable discrep-ancies between the behavior of Gb- and Gg-deficientmutants, including the double agg1 agg2 mutant. AGB1gene expression patterns in reproductive organs donot match AGG1, AGG2, or their combination. Hypo-cotyl elongation, both in darkness and under light, aswell as hook development are altered in the Gb-deficient mutant but not in any of the single Gg-deficient mutants or in the double agg1 agg2 mutant.Furthermore, agb1-2 was hypersensitive to ABA dur-ing germination, while all Gg-deficient mutants dis-played wild-type sensitivity. Striking discrepancieswere observed in the morphological and developmen-tal phenotypic characterization of the mutants. It isremarkable that none of the aerial morphological traitsfor which the Gb-deficient mutants show statisticallysignificant differences from the wild type can beobserved in the different Gg-deficient mutants. Theonly instance in which Gg1-deficient mutants, and thedouble agg1 agg2 mutant, show statistically significantdifferences from the wild-type controls is in floweringtime; however, this effect was opposite to that ob-served in the Gb-deficient mutant (Table I). Finally, ingpa1 (Wang et al., 2001; Coursol et al., 2003; Mishraet al., 2006) and agb1 (L.M. Fan, unpublished data),stomatal opening (but not stomatal closure) exhibitshyposensitivity to inhibition by ABA. In contrast, no

Table II. Silique morphology of gpa1 mutants and corresponding wild-type control plants

*, P , 0.05. ***, P , 0.001.

Characteristic Col-0 gpa1-3 gpa1-4 Ws gpa1-1 gpa1-2

Length (mm) 13.8 6 0.3 12.4 6 0.4* 12.7 6 0.3* 13.1 6 0.4 13.9 6 0.4 14.1 6 0.5*Blunt tip No Yes Yes No No NoPeduncle length (mm) 7.8 6 0.3 13.0 6 0.5*** 13.2 6 0.3*** 8.3 6 0.4 16.2 6 0.7*** 15.4 6 0.5***





ABA hyposensitivity of either ABA inhibition of sto-matal opening or ABA promotion of stomatal closurewas observed in any of the single Gg-deficient mutantsor the double agg1 agg2 mutant. Stomatal apertureresponses result from a complex web of cellular sig-naling events that regulate multiple effectors (Li et al.,2006). Therefore, we also assessed one particularlywell-defined subcellular event: ABA inhibition of in-ward K1 channels in guard cells (Blatt, 1990; Schwartzet al., 1994). In Ga- and Gb-deficient lines, the re-sponse of inward K1 currents to ABA is abrogated(Wang et al., 2001; Coursol et al., 2003; L.M. Fan,unpublished data). However, in all of the Gg-deficientmutants, guard cell K1 channel regulation by ABA waspresent at the same magnitude as in the wild type.Taken as a whole, our results demonstrate that theabsence of the Gg subunits does not completely phe-nocopy the lack of the Gb subunit.

To explain the observed discrepancies, two notnecessarily exclusive hypotheses could be proposed.The first is that additional Gg subunits exist inArabidopsis, which could possibly be expressed in re-productive organs to explain the expression discrep-ancies observed between AGB1 and the two known Ggsubunit genes, AGG1 and AGG2. Although exhaustivesearch of the fully sequenced Arabidopsis genome didnot reveal any additional canonical Gg subunits, thesesubunits display poor sequence conservation; there-fore, the presence of atypical subunits cannot bediscarded. This hypothesis allows retention of theheterotrimeric G protein dogma, developed mainlyfor mammalian systems, which states that all subunits

are interdependent and required for proper signalingof the heterotrimer (Gilman, 1987).

A second hypothesis predicts some functional au-tonomy of the G protein subunits in plants, consistentwith several recent observations. In animals, interde-pendence of Ga subunits and the corresponding Gbgdimers for correct subcellular localization has beenestablished (Takida and Wedegaertner, 2003), while inplants, the subunits do not depend on each otherfor plasma membrane targeting to the same extent(Adjobo-Hermans et al., 2006; Zeng et al., 2007; Wanget al., 2008). Moreover, both Arabidopsis Gg subunitslocalized to the plasma membrane in Ga- and Gb-deficient mutants (Zeng et al., 2007). Other unusualproperties of the plant G protein complex recently ledTemple and Jones (2007) to suggest that the plantheterotrimer is lagging behind in evolutionary termsfrom its animal counterparts. This antiquity couldallow some functional autonomy for the individualsubunits, since it is logical to propose that the hetero-trimer most probably originated from three initiallyindependent proteins. It is possible, therefore, that insome processes, plant Ga and Gb subunits might actindependently from each other and from Gg subunits.Notably, some Gg-dependent traits, such as suscepti-bility to necrotrophic fungi, methyl jasmonate sensi-tivity during root elongation and seed germination,and lateral root number, were altered in an oppositeway in gpa1-4 compared with agb1-2 (Trusov et al.,2006, 2007). At the same time, many traits reportedhere, including ABA sensitivity in seed germinationand stomatal regulation, floral organ shape, and hy-

Figure 5. ABA regulates stomatal movementssimilarly in Col and agg1-1c, agg2-1, agg2-2,and agg1 agg2. A, ABA (50 mM) inhibition oflight-induced stomatal opening in Col, agg1-1c,agg2-1, agg2-2, and agg1 agg2. Data shown aremeans 6 SE from three replicates with n . 150stomata for each experiment. B, ABA (50 mM)induction of stomatal closure in Col, agg1-1c,agg2-1, agg2-2, and agg1 agg2. Data shown aremeans 6 SE from three replicates with n . 150stomata for each experiment. C, ABA (20 mM)inhibition of light-induced stomatal opening inCol, agg1-1c, agg2-1, agg2-2, and agg1 agg2.Data shown are means 6 SE from three repli-cates with n . 150 stomata for each experi-ment. D, ABA (20 mM) induction of stomatalclosure in Col, agg1-1c, agg2-1, agg2-2, andagg1 agg2. Data shown are means 6 SE fromthree replicates with n . 150 stomata for eachexperiment.

Trusov et al.




pocotyl elongation in darkness, which were similarlyaltered in gpa1-4 and agb1-2 mutants, were not alteredin Gg mutants. This could imply that Ga and Gb, butnot always Gg, are part of an as yet uncharacterizedcomplex, which governs those traits. Interestingly, inrice, all individual heterotrimeric G protein subunitswere found as a part of 400-kD multiprotein com-plexes, but a fraction of the Gbg dimers were alsofound not to be associated with those complexes (Katoet al., 2004). In Arabidopsis, membrane-associated 400-kD protein complexes were reported for the ERECTAreceptor-like kinase (Shpak et al., 2003), and knockoutsof ERECTA and ERECTA-like genes display similarphenotypes to Ga- and/or Gb-deficient mutants in anumber of traits (Lease et al., 2001; Shpak et al., 2004)as well as increased susceptibility to necrotrophicpathogens (Llorente et al., 2005), suggesting that G pro-teins could be an integral part of those complexes.Moreover, during the preparation of this article, it wasreported that in Arabidopsis roughly 30% of the nativeGa and all of the overexpressed cyan fluorescent

protein (CFP)-Gb are associated with large (approxi-mately 700 kD) multiprotein complexes found in theplasma membrane fraction (Wang et al., 2008). Unfor-tunately, no information on Gg-containing complexeswas provided for either of the Gg subunits (Wanget al., 2008).

The question arises whether plant Gb can act inde-pendently of Gg, as has already been described forGb5 in animal systems. The mammalian Gb5 subunitcan form functional dimers with a number of RGSproteins that contain a ‘‘Gg-like motif’’ known as theGGL domain, precluding formation of the canonicaldimer Gb5g (Snow et al., 1998; Witherow et al., 2000;Sondek and Siderovski, 2001; Witherow and Slepak,2003; Willars, 2006). There are no known GGL domain-containing plant proteins; reports about plant GGLdomain proteins, however, are currently available. Inaddition, the WD40 propeller structure of the Gbsubunit allows interaction with multiple proteins,hence providing a scaffold for large protein assem-blies. In Arabidopsis and tobacco (Nicotiana tabacum),

Figure 6. ABA regulates K1 currents similarly in guard cells of Col, agg1-1c, agg2-1, agg2-2, and agg1 agg2. A, Typical wholecell recordings of guard cell K1 currents with or without 50 mM ABA. Time and voltage scales shown in the top right panel applyto all panels. B, Current/voltage relationship (mean 6 SE) of time-activated whole cell K1 currents as illustrated in A. Number ofguard cells was as follows: Col (11), Col 1 ABA (13), agg1-1c (10), agg1-1c 1 ABA (10), agg2-1 (eight), agg2-1 1 ABA (16), agg2-2(six), agg2-2 1 ABA (seven), agg1 agg2 double mutant (16), and agg1 agg2 double mutant 1 ABA (17).





the Gb subunit was identified in plasma membraneTriton X-100-insoluble microdomains along with othersignaling components, including kinases and smallGTP-binding proteins (Peskan and Oelmuller, 2000;Peskan et al., 2000; Shahollari et al., 2004). Thus, it isnot unlikely that Gb could function independentlyfrom Gg as an integral part of multiprotein complexesto mediate some processes while functioning as a Gbgdimer in others. In this scenario, knockout of AGB1would destroy both the Gbg dimers and the hypo-thetical Gb-containing complexes, while the absenceof Gg subunits would only obliterate signaling thatwas directly dependent on Gbg dimers.

In both hypotheses, it is necessary to explain howGb can be targeted to the plasma membrane in theabsence of both Gg subunits. A hybrid CFP-AGB1fusion protein showed diffuse localization in the cy-toplasm unless coexpressed with either AGG1 orAGG2 subunits (Adjobo-Hermans et al., 2006). Thisstudy demonstrated sufficiency of the Gg subunits forthe plasma membrane localization of Gb. However,since this study was based on high ectopic expressionof the AGB1, AGG1, and AGG2 genes, it is possible thatother proteins, or complexes, can also anchor Gb to theplasma membrane under normal circumstances. Inthis respect, it will be interesting to determine whetherAGB1 is membrane localized in the agg1 agg2 doublemutant. Just as for all other known Gg subunits, theability of AGG1 and AGG2 to be targeted to theplasma membrane crucially relies on prenylation ofthe C-terminal CAAX motif (Zeng et al., 2007). InArabidopsis, two prenylation enzymes, geranylgera-nyltransferase I (PGGT-I) and farnesyltransferase (PFT),have been identified (Caldelari et al., 2001; Runninget al., 2004). Interestingly, knockout of PFT results inABA hypersensitivity and phenotype alterations sim-ilar to those observed in Ga- and Gb-deficient mu-tants, while knockout of PGGT-I, which has beenshown to prenylate both AGG1 and AGG2 subunits(Zeng et al., 2007), leads to wild-type phenotype andwild-type ABA sensitivity in seed germination (Johnsonet al., 2005; Zeng et al., 2007). Taken together withthe fact that knockout of both Gg subunits does notcompletely phenocopy Gb-deficient mutant pheno-types, these observations suggest that there are addi-tional, probably farnesylated, proteins interacting withAGB1 in Arabidopsis. It is possible, therefore, thatcurrently unknown Gg subunits, GGL-containingRGSs, or some components of the multiprotein com-plexes described by Wang et al. (2008) can be pre-nylated by PFT and are able to target AGB1 to theplasma membrane. It is interesting that in rice, the Ggsubunit RGG2 lacks the C-terminal prenylation motifbut nevertheless is detected in the plasma membranefraction associated with Gb (Kato et al., 2004).

CONCLUSION

Additional studies will prove or disprove the feasi-bility of the hypotheses described above. Neverthe-

less, whatever the result of those studies, our dataunambiguously reveal that the variety of heterotri-meric G proteins in plants is not limited to the twocanonical heterotrimers Gabg1 and Gabg2 found sofar. This adds an additional level of complexity to themolecular mechanisms used by G proteins in plantsand provides a new degree of functional selectivity tothat reported previously for the two known hetero-trimers (Trusov et al., 2007).

MATERIALS AND METHODS

Plant Material

The Arabidopsis (Arabidopsis thaliana) agg1-1c mutant allele of AGG1

(At3g63420), the agg2-1 mutant allele of AGG2 (At3g22942), the double mutant

agg1 agg2, and the agb1-2 mutant were described previously (Ullah et al., 2003;

Trusov et al., 2007). New alleles agg1-2 and agg2-2 (both in the Col-0

background) were produced as T-DNA mutants by GaBI-Kat (Rosso et al.,

2003). agg1-2 seeds were obtained from GaBI-Kat (accession no. 736A08),

while agg2-2 lines were obtained from the Nottingham Arabidopsis Science

Centre (accession no. N375172). For each new line, homozygous plants were

selected using a three-primer PCR approach. The exact position of the T-DNA

insertion was determined by amplifying and sequencing a genomic DNA

fragment between the T-DNA end and the 3# end of the gene in the

chromosome. Absence of full-length AGG2 mRNA for agg2-2 was confirmed

by reverse transcription (RT)-PCR (data not shown). In agg1-2, the insert is

located in the promoter region, and full-size AGG1 mRNA was detected in the

mutant. However, northern analysis revealed significant down-regulation in

AGG1 mRNA levels in mutant plants (roughly 30% of the wild-type level;

Supplemental Fig. S1).

Mutant Characterization

Mature plants were grown under a long-day (16 h of light/8 h of dark) and

23�C regimen for 6 weeks, and an additional 2 weeks were allowed for seed

maturation. Morphological characteristics were measured at defined devel-

opmental stages as described elsewhere (Boyes et al., 2001). For destructive

analysis (flower, silique, and leaf shape traits), plants were removed from the

rest of the population randomly, dissected, and photographed if necessary.

Seed and Seedling Assays

All plates contained 0.53 Murashige and Skoog basal salts (PhytoTechnol-

ogy Laboratories) and 0.8% agar. Stock solutions of ABA at the designated

concentrations were added to autoclaved medium cooled to approximately

55�C. Since germination is extremely sensitive to the growth conditions

experienced by the parental plant and to postharvest storage, all seed lots for

seed and seedling assays were collected at the same time from plants grown

simultaneously under the same conditions. The seeds were stored at 4�C in the

dark. Seeds were dry sterilized by 3 h of incubation in a chamber filled with

chlorine gas. Approximately 150 sterilized seeds of all tested lines were

planted on the same petri dish with a designated treatment. After sowing, all

seeds were stratified for at least 48 h at 4�C in darkness. Germination was

defined as an obvious protrusion of the radicle.

For hypocotyl elongation assays, seeds were induced under continuous

light (150 mmol m22 s21) for 24 h, then seedlings were grown on vertical plates

for 1 to 3 d in a dark cabinet. The plates were photographed and hypocotyl

length was measured.

Isolation of RNA and Transcript Analysis

Total RNA for northern analysis and RT-PCR was extracted as described

previously (Trusov and Botella, 2006; Purnell and Botella, 2007). Probes for

northern blots were labeled using a Rediprime II P32 radiolabeling kit

(Amersham). Membranes were hybridized overnight in Church buffer

(Church and Gilbert, 1984) at 65�C, washed twice in 0.1% SSC and 0.1% SDS

solution as described (Petsch et al., 2005), and exposed to PhosphorImager

Trusov et al.




plates for analysis (Molecular Dynamics). For RT-PCR, reverse transcriptions

were carried out using the SuperScript III RT kit according to the manufac-

turer’s instructions (Invitrogen) as described previously (Moyle et al., 2005).

PCR amplifications were performed using GoTaq Green Master mix (Promega)

in 35 cycles with the following parameters: 94�C for 30 s, 54�C for 30 s, and

72�C for 1 min. The primers used for the AGG1 and AGG2 genes were de-

scribed previously (Trusov et al., 2007).

Stomatal Aperture Experiments and GuardCell Electrophysiology

Arabidopsis plants were grown in soil mix (Potting Mix; Miracle-Gro) in

growth chambers with 8-/16-h light/dark and 22�C/20�C cycles. Fully

expanded young leaves from 4-week-old plants were used for both stomatal

aperture assays and guard cell protoplast isolation. All of the protocols for

stomatal aperture assays and whole cell K1 current recordings from guard

cells with and without ABA treatment were as described for previous analyses

of Ga-deficient mutant plants (Wang et al., 2001; Coursol et al., 2003). For

whole cell K1 current analysis, recordings obtained at 10 min after formation

of the whole cell configuration were used and were analyzed as described

(Coursol et al., 2003). Whole cell capacitances were used to normalize current

amplitude (pA/pF) to avoid the influence of cell size. Data were compared

with Student’s t test, and results with P , 0.01 were considered significantly

different.

Supplemental Data

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

Supplemental Figure S1. Down-regulation of AGG1 gene expression in

agg1-2 T-DNA mutants.

ACKNOWLEDGMENTS

We thank Dr. Mike Mason and Dr. David Chakravorty for critical reading

of the manuscript.

Received February 13, 2008; accepted April 22, 2008; published April 25, 2008.

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