Gg1 1 Gg2 Gb: Heterotrimeric G Protein Gg-Deficient
Mutants Do Not Recapitulate All Phenotypes of
Gb-Deficient Mutants1[C][W][OA]
Yuri Trusov, Wei Zhang, Sarah M. Assmann, and José Ramón Botella*
Plant Genetic Engineering Laboratory, Department of Botany, School of Integrative Biology, University of
Queensland, Brisbane, Queensland 4072, Australia (Y.T., J.R.B.); and Biology Department, Pennsylvania
State 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 a
functional Gg subunit for active signaling predicts that a mutant lacking both AGG1 and AGG2 proteins should phenotypically resemble mutants lacking AGB1 in all respects. We previously reported that Gb- and Gg-deficient mutants coincide
during 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 lacking
both Gg subunits. While Gb-deficient mutants are hypersensitive to abscisic acid inhibition of seed germination and are
hyposensitive to abscisic acid inhibition of stomatal opening and guard cell inward K1 currents, none of the available Ggdeficient mutants shows any deviation from the wild type in these responses, nor do they show the hypocotyl elongation and
hook development defects that are characteristic of Gb-deficient mutants. In addition, striking discrepancies were observed in
the 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 tight
relationship between Gb and Gg subunits in plants, considering the significant differences reported here, we hypothesize the
existence of new and as yet unknown elements in the heterotrimeric G protein signaling complex.
Heterotrimeric G proteins contain Ga, Gb, and Gg
subunits and transduce signals from activated plasma
membrane receptors to intracellular effectors (Gilman,
1987). Upon activation of the receptor, GDP bound to
inactive Ga is exchanged for GTP, causing a conformational change that leads to activation with or without physical dissociation of the Ga subunit from the
Gbg 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 transmit the signal to their specific effector molecules until
intrinsic GTPase activity of the Ga subunit hydrolyzes
the GTP molecule, thus returning Ga to its inactive state and sequestering Gbg back to the inactive
1
This work was supported by the Australian Research Council
(Discovery Grant nos. DP0344924 and DP0772145), the U.S. Department of Agriculture (grant no. 2006–35100–17254), and the National
Science Foundation (grant no. MCB–0209694).
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
José Ramón Botella ([email protected]).
[C]
Some figures in this article are displayed in color online but in
black 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 subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.117655
636
heterotrimer. Gb and Gg subunits form tightly bound
dimers that work as functional units and can only be
dissociated under strong denaturing conditions
(Schmidt et al., 1992; Clapham and Neer, 1993; Gautam
et al., 1998; McCudden et al., 2005).
In animal systems, G proteins mediate the signaling
of 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 the
three subunits, and different combinatorial possibilities provide the required specificity for multitudinous G protein-based signaling pathways (Gautam
et al., 1998; Balcueva et al., 2000; Wettschureck and
Offermanns, 2005; Marrari et al., 2007). In open contrast, plants only contain one or two genes for each of
the subunits (Ma et al., 1990; Poulsen et al., 1994; Weiss
et al., 1994; Gotor et al., 1996; Iwasaki et al., 1997; Seo
et 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; Kato
et al., 2004; Misra et al., 2007). In Arabidopsis (Arabidopsis thaliana), a single Ga (Ma et al., 1990; Ma, 1994), a
single Gb (Weiss et al., 1994), and two Gg (Mason and
Botella, 2000, 2001) subunit genes have been identified.
Despite the fact that only two combinatorial possibilities are feasible for the heterotrimers in Arabidopsis, G proteins are involved in multiple processes
(Assmann, 2004; Jones and Assmann, 2004; PerfusBarbeoch et al., 2004). Recent genetic studies using
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Heterotrimeric G Proteins in Plants: Gg Subunits
Ga- and Gb-deficient or -overproducing mutants have
demonstrated the involvement of G proteins in abscisic acid (ABA) and brassinosteroid sensitivity during
seed germination and early plant development (Ullah
et al., 2002; Lapik and Kaufman, 2003; Chen et al., 2004,
2006; Pandey et al., 2006; Warpeha et al., 2006), stomatal 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 (Lease
et al., 2001; Ullah et al., 2003), plant defense against
necrotrophic 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 has
been 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 functional
Gg subunit is a compulsory prerequisite for the functioning of the entire heterotrimer, and lack of Gg
subunits results in the obliteration of both Gbg- and
Ga-mediated pathways (Gilman, 1987; Kisselev et al.,
1994; Manahan et al., 2000; Krystofova and Borkovich,
2005; Myung et al., 2006). There is one notable exception to this rule: the human neurospecific Gb5 subunit
forms functional dimers with some members of the
regulator of G protein signaling (RGS) subfamily C
proteins instead of with conventional Gg subunits
(Snow et al., 1998; Sondek and Siderovski, 2001). Plant
G proteins appear to behave similarly to their animal
counterparts, and tight physical interaction between
Gb and each of the Gg subunits has been demonstrated in vitro (Mason and Botella, 2000, 2001) and in
vivo (Kato et al., 2004; Adjobo-Hermans et al., 2006).
Moreover, it has been shown that Arabidopsis Gb
subunit localization on the plasma membrane requires
a Gg subunit (Obrdlik et al., 2000; Adjobo-Hermans
et al., 2006). Evidence for functional interaction of the
subunits was recently provided using overexpression
of a truncated Gg1 subunit lacking the isoprenylation
motif (which is responsible for anchoring the bg dimer
to the membrane) and mutants lacking each of or both
Gg subunits (Chakravorty and Botella, 2007; Trusov
et al., 2007). It has also been shown that different Gg
subunits confer specificity to the Gbg dimer, with the
Gbg1 dimer mediating signal transduction events
during plant defense against necrotrophic fungi,
acropetal auxin signaling in roots, and osmotic stress
regulation of seed germination, while Gbg2 is involved in basipetal auxin signaling in roots and D-Glc
sensing during germination (Trusov et al., 2007).
However, despite the above-mentioned similarities
between plant and animal G proteins, a number of
unusual properties observed in the plant subunits
have led to suggestions that, in some cases, the animal
paradigm may not necessarily hold true in plants. For
instance, it was established that unlike animal subunits, Arabidopsis Ga and Gbg are capable of tethering 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 shown
that the GTPase activity of the Arabidopsis Ga subunit
is very low, leading to the hypothesis by some authors
that Ga might be in the activated state by default
(Willard and Siderovski, 2004; Johnston et al., 2007a;
Temple and Jones, 2007). In addition, the interaction
between Ga and the Gbg dimer in rice has been
reported to be relatively weak compared with that in
animal systems (Kato et al., 2004), although formation
of the heterotrimer in vivo has been demonstrated
(Kato et al., 2004; Adjobo-Hermans et al., 2006). Recently, it was shown that in Arabidopsis both Ga and
Gb are associated with large macromolecular complexes of approximately 700 kD (Wang et al., 2008).
Finally, Arabidopsis Gg subunits are capable of being
targeted to the plasma membrane in mutants lacking
functional Ga and Gb subunits (Zeng et al., 2007).
Here, we present data conflicting with the established heterotrimeric G protein model. We found that a
number of phenotypic alterations observed in Ga- or
Gb-deficient mutants cannot be detected in mutants
lacking Gg1, Gg2, or both Gg subunits. Our results
raise the possibility that Gg subunits are not required
for some Ga- and Gb-mediated processes in Arabidopsis or that additional nonconventional Gg subunits
exist in this species.
RESULTS
The Expression Profiles of AGG1 and AGG2 in
Reproductive Organs Do Not Match the AGB1
Expression Pattern
We previously reported that GUS staining patterns
in transgenic Arabidopsis ecotype Columbia (Col-0)
plants carrying promoter fusions of each of the AGG1
and AGG2 genes with the GUS reporter gene were
tissue specific and that together they overlap AGB1
expression patterns in most plant tissues and developmental stages (Anderson and Botella, 2007; Trusov
et al., 2007). Nevertheless, a number of small but
important differences can also be observed, especially
in reproductive tissues. Analysis of GUS expression in
flowers and siliques revealed that AGB1 is moderately
expressed 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 observed at both ends, gradually disappearing toward
the center (Fig. 1D). In AGG1:GUS plants, GUS expression was only detected in the stigma of mature
flowers (Fig. 1, B and C), possibly correlating with
pollination, while in siliques only the abscission zone
showed staining (Fig. 1D). In AGG2:GUS transformants, GUS expression was evident in the apex of
stamen filaments at a very early developmental stage
and disappeared before the flower opened (Fig.
1C). No GUS staining was detected in siliques of
AGG2:GUS plants. Discrepancies were also observed
in germinating seeds, where distinct GUS staining in
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Trusov et al.
Figure 1. AGB1, AGG1, and AGG2 expression patterns in flowers.
Histochemical analysis of GUS expression in transgenic Arabidopsis
plants carrying AGB1:GUS, AGG1:GUS, or AGG2:GUS fusions. A,
Fully opened flowers. B, Stigma of young (unopened bud) and mature
flowers. C, Anther, stamen filaments, and pollen. D, Green-stage
siliques. E, Germinating seedlings at 24, 48, and 96 h after imbibition.
AGB1:GUS plants was observed much earlier (24 and
48 h after imbibition) than in AGG1:GUS or AGG2:GUS
plants. However, expression in AGG1:GUS or AGG2:
GUS increased to detectable levels and overlapped
with AGB1 expression in 4-d-old seedlings (Fig. 1E), in
agreement with a previous report (Trusov et al., 2007).
ABA Sensitivity of Gg1- and Gg2-Deficient Mutants
in Germination
Heterotrimeric G protein involvement in different
aspects of seed germination has been established by a
number of studies (Ullah et al., 2002; Lapik and
Kaufman, 2003; Chen et al., 2004, 2006; Pandey et al.,
2006; Liu et al., 2007; Warpeha et al., 2007). Involvement
of the heterotrimeric G protein signaling components
GPA1, AGB1, GCR1, and RGS1 in ABA inhibition of
seedgermination is well documented (Ullah et al., 2002;
Chen et al., 2004, 2006; Pandey et al., 2006). It was
recently proposed that a second putative G proteincoupled receptor (GCR2) is a plasma membrane receptor for ABA (Liu et al., 2007); however, these results
have been challenged (Gao et al., 2007; Johnston et al.,
2007b; Illingworth et al., 2008).
Roles for the Arabidopsis Gg subunits, AGG1 and
AGG2, in D-Glc and osmotic sensing during germination were recently reported (Trusov et al., 2007), but no
data are available at present on their involvement in
ABA sensing. Therefore, we compared the responses
of Ga-, Gb-, and Gg-deficient mutants to ABA-mediated
inhibition of germination (Fig. 2). We analyzed germination rates for seven different seed lots for each
genotype (stored in an identical environment for approximately 1 month after harvest) under a number of
experimental conditions. Each lot was tested at least
twice. In all tests, all genotypes showed 100% germination on control medium (no ABA added) by day 3
after light exposure (data not shown). In accordance
with previous reports showing hypersensitivity of
Ga- and Gb-deficient mutants to ABA during germination (Pandey et al., 2006), gpa1-4 and agb1-2 mutants
showed enhanced ABA-mediated inhibition of germination compared with the wild type (Fig. 2, A and B).
In contrast, four independent single Gg-deficient mutants, agg1-1c, agg1-2, agg2-1, and agg2-2, as well as the
double agg1 agg2 mutant showed levels of ABA sensitivity 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 decreased or slightly increased ABA sensitivity compared with the wild type, but the responses never
reached the hypersensitivity levels displayed by Gbdeficient mutants. Figure 2, A and B, shows germination rates for the wild type and all mutants in a
representative experiment. The differences in ABA
sensitivity between gpa1-4 and agb1-2 mutants and all
of the other genotypes analyzed (the wild type and
Gg-deficient mutants) were statistically significant
(P , 0.05).
It is known that high sugar concentration inhibits germination, while low amounts of Suc or Glc
can rescue ABA-mediated inhibition of germination
(Garciarrubio et al., 1997; Price et al., 2003). We analyzed germination rates on ABA-containing medium
in the presence or absence of 2% Suc. Suc increased the
germination of all genotypes at the two ABA concentrations assayed (Fig. 2, A and B), although, due to the
complexity of the figure, it is difficult to visualize and
compare the effects of Suc in the different genotypes.
One useful way to visualize differences in behavior is
to plot the relative effect of Suc on germination inhibition by ABA (Fig. 2C). Of the two ABA concentrations studied, only one (5 mM ABA) is amenable to this
type of analysis, since in these specific experimental
conditions, the percentage of germinated seeds
showed a linear increase during the studied period
(3–6 d after induction). In contrast, using 2 mM ABA,
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Heterotrimeric G Proteins in Plants: Gg Subunits
Figure 2. Sensitivity to ABA-induced inhibition of seed germination in G protein complex mutants.
A and B, Seeds from matched seed
lots were surface sterilized and
plated on 0.53 Murashige and
Skoog medium plates in the presence of 2 mM (A) or 5 mM (B) ABA.
Plates were transferred to 100 mmol
m22 s21 light and 23°C. Germination was recorded at 3, 4, 5, and 6 d
after transfer of the plates under light
and expressed as a percentage of
total number of seeds. The experiment was repeated three times, and
data were averaged (n . 100 for
each experiment). The error bars
represent SE. C, Rescue effect of
Suc on germination inhibited by
5 mM ABA. Error bars indicate SE
values obtained from four measurements. Asterisks indicate values statistically significantly different from
the wild type (P , 0.05).
almost 100% germination was observed on days 5 to 6
for most genotypes. This allowed us to calculate the
‘‘rescue’’ effect of Suc as the relative increase in germination [e 5 (s 2 n)/s, where s is the percentage of
germinated seeds on Suc-containing plates and n is the
percentage of germinated seeds on plates without Suc]
and average it for the four time points. Both gpa1-4
and agb1-2 mutants showed statistically significantly
higher values than the remaining genotypes (P , 0.05),
although absolute germination levels remained significantly (P , 0.05) lower for these genotypes than for all
others.
Gg-Deficient Mutants Do Not Display the agb1-2
Deetiolated Phenotype in Darkness
Partially deetiolated seedlings of Ga- and Gb-deficient
mutants have short hypocotyls with visibly increased
girth and a characteristic open hook (Ullah et al., 2001,
2003; Wang et al., 2006). We analyzed hypocotyl elongation rate and hook development in darkness as well
as 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 synchronized germination, all seeds were stratified for 5 d
and induced to germinate under 150 mmol m22 s21
continuous light during 24 h. Afterward, plates with
seeds were placed vertically either in a dark cabinet or
under continuous light (90 mmol m22 s21) for 24, 48,
and 72 h.
Figure 3, A and B, shows hypocotyl elongation
dynamics of all mutant genotypes and the wild type
grown in darkness and light, respectively. In agreement with previous reports (Ullah et al., 2001, 2003),
hypocotyl elongation rates for Ga- and Gb-deficient
mutants (gpa1-4 and agb1-2) in darkness were lower
compared with those for the wild type, with statistically significant differences after 24 and 48 h (P , 0.01
and P , 0.05, respectively). Under light, gpa1-4 and
agb1-2 hypocotyls were significantly shorter than wildtype hypocotyls at all three time points (at least P ,
0.05). In open contrast to the behavior of gpa1-4 and
agb1-2 mutants, hypocotyl elongation in either dark or
light conditions in each of the individual Gg1- or Gg2deficient mutants or the double agg1 agg2 mutant was
not statistically different from that in the wild type.
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Trusov et al.
Figure 3. Seedling development of G protein complex mutants grown in darkness or under light. A and
B, Hypocotyl elongation rates of the wild type and the
mutants at 24, 48, and 72 h after germination in
darkness (A) or under 90 mmol m22 s21 light (B). Error
bars indicate SE. At least 20 seedlings were measured.
C, Degree of hook opening in dark-grown wild-type
and mutant seedlings measured approximately 24 h
after germination. SE values indicated by error bars are
based on a minimum of 20 seedlings. Closed hooks
were treated as having zero degree of opening. Triple
asterisks indicate values statistically significantly different from the wild type (P , 0.001). The inset shows
representative seedlings in order from left to right:
Col-0, gpa1-4, agb1-2, agg1-1c, agg1-2, agg2-1,
agg2-2, and agg1 agg2.
When the hook angle was measured after 24 h of
dark incubation, gpa1-4 and agb1-2 mutants showed
the typical ‘‘open-hook’’ phenotype described previously (Ullah et al., 2003; Fig. 2C). In contrast, all of the
single Gg-deficient mutants as well as the double agg1
agg2 mutant showed a wild-type phenotype clearly
different from those of gpa1-4 and agb1-2.
None of the Morphological Alterations of Aerial Organs
Observed in Ga- and Gb-Deficient Mutants Is Present in
Gg-Deficient Mutants
Phenotypic analyses of Ga- and Gb-deficient mutants have revealed a number of developmental and
morphological abnormalities (Ullah et al., 2003). In
order 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 agg1
agg2 grown under ‘‘standard’’ conditions as specified
in ‘‘Materials and Methods.’’ Quantitative analysis of a
number of morphological characteristics was carried
out at defined growth stages (Boyes et al., 2001) during
development (Table I; Fig. 4). Mean values of these
traits in the mutant lines were analyzed for significant
deviation from the corresponding values in the wild
type by pair-wise, two-sample Student’s t test. Unless
stated otherwise, the mean values were derived from
analysis of at least 30 plants grown in a checkerboard
pattern.
Differences between agb1-2 and wild-type plants
become apparent from a very early stage of development. Smaller and rounder cotyledons of agb1-2 mu-
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Heterotrimeric G Proteins in Plants: Gg Subunits
Figure 4. Comparison of characteristic morphological
traits 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, Fully
open mature flowers. D, Fully expanded siliques. [See
online article for color version of this figure.]
tants are apparent as early as 7 d after germination
(Fig. 4A). The shape of the gpa1-4 cotyledons is very
similar to that of wild-type cotyledons at this stage,
although they are larger (Fig. 4A). In contrast, all of the
single agg1-1c, agg1-2, agg2-1, and agg2-2 mutants as
well as the double agg1 agg2 mutant are indistinguishable from the wild type at this developmental stage
(Fig. 4A). Measurements of at least 50 seedlings of each
genotype revealed that these visible differences between agb1-2 or gpa1-4 and the wild type are statistically significant (P , 0.001), while all Gg-deficient
mutants were indeed similar to the wild type (Table I).
Rosette diameter measured at the inflorescence emergence 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 early
in Gb-deficient mutants, giving a distinctive appearance caused by the cotyledons being very close to the
hypocotyl (Fig. 4A). The rosette leaves of gpa1-4 and
agb1-2 mutants have a characteristic crinkled surface
and rounder appearance compared with those of the
wild type (Fig. 4B). The ratios between leaf length and
width in Ga- and Gb-deficient mutants were significantly lower than in the wild type (P , 0.001; Table I).
In contrast, neither the individual Gg-deficient mutants nor the double agg1 agg2 mutant showed any
statistically significant differences from the wild type
for any of the above-mentioned traits: rosette diameter,
leaf appearance, petiole size, and leaf length-width
ratio.
Inflorescence emergence, defined by the appearance
of flower buds, occurs approximately 2 d earlier in
agg1-1c, agg1-2, and double agg1 agg2 mutants than
in the wild type and gpa1-4, agg2-1, and agg2-2 mutants. 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 inflorescence emergence (P , 0.05), the first agb1-2 flower
opened at the same time as for the wild type, gpa1-4,
and Gg2-deficient mutants (data not shown). The final
inflorescence height was noticeably lower in agb1-2 (P
, 0.01), while all other mutants were similar in height
to the wild type. Apical dominance is known to be
regulated by basipetal auxin flow from the apical
meristem (Jones, 1998; Dun et al., 2006; Leyser, 2006).
Attenuation of auxin signaling by Gb and both Gg
subunits has been established previously in roots
(Ullah et al., 2003; Trusov et al., 2007). In the floral
stem, our data showed increased apical dominance in
agb1 mutants, as evidenced by a decreased number of
branches, which is consistent with previous reports
(Ullah et al., 2003). In contrast, all of the Gg-deficient
mutants showed a wild-type floral stem branching
pattern. Contrary to a previous report (Ullah et al.,
2003), we found that the number of open flowers at the
midflowering 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 gpa1-4
mutants, while all Gg-deficient mutants had flowers of
similar size compared with the wild type (Table I; Fig.
4C). Even though we measured only the diameter of
the fully open flower, sepal size was also affected in
agb1-2 and gpa1-4 mutants, as can be seen in Figure 4C.
Alteration of the silique shape was first described
for the agb1-1 mutant (Lease et al., 2001), and similar
observations were made for agb1-2 (Ullah et al., 2003).
Our measurements of agb1-2 concur with previous
reports 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 produced shorter (although to a lesser extent) and wider
siliques, with the characteristic blunt tip. Curiously,
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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)
Rosette/leaf
Length-width ratio
Petiole length (mm)
Crinkly surface
Rosette diameter (mm)
Inflorescence/flowers
Buds are visible (day)
Length (cm)
No. of branches
No. of open flowers
Flower diameter (mm)
Silique
Length (mm)
Width (mm)
Blunt tip
Peduncle length (mm)
No. of seeds
No. of siliques (per cm)
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
3.90
3.90
3.90
3.90
1.8 6 0.07
9.6 6 0.4
No
41.3 6 1.1
1.3 6 0.05***
10.8 6 0.3**
Yes
43.0 6 1.3
1.2 6 0.04***
6.7 6 0.3***
Yes
29.2 6 1.2***
1.9 6 0.07
9.3 6 0.3
No
44.5 6 1.0
1.8 6 0.06
9.4 6 0.4
No
41.4 6 1.2
1.8 6 0.08
8.9 6 0.5
No
43.6 6 1.0
1.8 6 0.09
9.7 6 0.3
No
43.9 6 1.3
1.7 6 0.12
8.8 6 0.4
No
40.8 6 1.8
5.10
6.90
6.90
6.50
6.50
22.7
33.6
3.4
3.2
4.0
23.1
30.1
3.5
3.4
3.5
24.9
26.6
2.4
6.4
3.3
18.3
32.2
3.1
3.2
4.1
19.3
31.2
3.2
2.9
4.2
22.4
32.3
3.5
2.9
4.4
21.9
31.2
3.4
3.1
4.3
19.8
32.3
3.1
3.5
4.0
6.90
6.90
6.90
6.90
8.00
9.70
13.8 6 0.3
0.73 6 0.02
No
7.8 6 0.3
51.6 6 1.3
0.88 6 0.02
6
6
6
6
6
1.1
0.6
0.4
0.2
0.3
6
6
6
6
6
1.3
1.6
0.6
0.3
0.1*
12.7 6 0.3*
0.82 6 0.03**
Yes
13.2 6 0.5***
44.0 6 1.2**
0.93 6 0.03
6
6
6
6
6
0.9*
1.3**
0.5*
1.6**
0.2*
10.6 6 0.2***
0.93 6 0.03***
Yes
9.9 6 0.3*
39.6 6 0.9***
1.13 6 0.02***
these results differ from those reported by Ullah et al.
(2003), who analyzed two different Ga-deficient mutants, gpa1-1 and gpa1-2, and found their siliques to be
slightly longer than wild-type siliques and having a
wild-type tip shape. It is noteworthy that gpa1-1 and
gpa1-2 mutants were obtained in the Wassilewskija
(Ws) ecotype, while gpa1-4 (as well as another Gadeficient mutant, gpa1-3) is in the Col-0 background.
To analyze the effects of growth conditions or ecotype
in silique development, we simultaneously grew
gpa1-1, gpa1-2, gpa1-3, and gpa1-4 mutants and corresponding wild-type plants. Interestingly, siliques of
gpa1-1 and gpa1-2 were indeed slightly longer than
those of the Ws wild type, with ‘‘normal’’ acute tips, as
described by Ullah et al. (2003), while both gpa1-3 and
gpa1-4 siliques were shorter than those in the Col-0
wild type, and both had blunt tips (Table II). This
observation is quite important, as it demonstrates that
some effects of GPA1 deficiency are dependent on the
genetic background and are not necessarily universal,
even within the same species. Siliques in all Ggdeficient mutants showed wild-type characteristics.
In contrast to all other aerial organs, in which the
knockout of AGB1 results in a reduced size, the silique
peduncle was longer in agb1-2 mutants (Table I), and
this trait was even more pronounced in gpa1-4 plants
(P , 0.001). As has been the case with most of the
morphological traits studied here, all Gg-deficient
mutants showed wild-type peduncle length (Table I).
It is worth mentioning that, even though Ga-deficient
mutants showed ecotype-dependent behavior for silique length and tip shape, the peduncles of gpa1-1,
gpa1-2, gpa1-3, and gpa1-4 were almost twice as long as
those of the relevant Ws or Col-0 wild-type control
plants (P , 0.001; Table II). Importantly, peduncle
length values presented here were obtained from the
first two to three (lowest) siliques per plant, as peduncle length decreases gradually in size toward the top of
the inflorescence. Nevertheless, the described trends
6
6
6
6
6
1.5*
1.4
0.5
0.6
0.1
13.9 6 0.4
0.72 6 0.02
No
7.7 6 0.3
55.3 6 1.0
0.95 6 0.02
6
6
6
6
6
0.8*
1.2
0.5
0.4
0.1
13.7 6 0.4
0.70 6 0.02
No
8.2 6 0.4
51.0 6 1.1
0.81 6 0.02
6
6
6
6
6
1.1
0.8
0.6
0.6
0.2
14.1 6 0.5
0.72 6 0.02
No
8.1 6 0.3
50.0 6 2.5
0.81 6 0.01
6
6
6
6
6
1.3
1.1
0.5
0.3
0.2
13.8 6 0.3
0.71 6 0.02
No
7.9 6 0.3
53.3 6 1.5
0.93 6 0.02
6
6
6
6
6
0.7*
1.8
0.5
0.4
0.1
13.2 6 0.3
0.72 6 0.02
No
8.1 6 0.3
49.3 6 1.8
0.94 6 0.04
were conserved along the entire inflorescence (data
not shown). The number of seeds per silique was
calculated using three siliques per plant and averaged
for 10 plants per genotype. Siliques of agb1-2 and gpa1-4
mutants contained fewer seeds than wild-type controls (P , 0.001 and P , 0.01, respectively), while none
of the Gg-deficient mutants showed statistically significant difference from the wild type. The shorter
inflorescence and higher number of siliques present in
agb1-2 mutant plants resulted in a highly statistically
significant difference in the density of siliques (number of siliques per centimeter of inflorescence), while
Ga- and all Gg-deficient mutants were similar to the
wild type (Table I).
Guard Cells of Gg-Deficient Mutants Show Wild-Type
Responses to ABA
ABA is a well-studied phytohormone in guard
cell signaling and is an important component of
many stress responses in plants. In Arabidopsis, Gadeficient mutants show alterations in a number of
guard cell responses, such as hyposensitivity to ABA
inhibition of stomatal opening and reduced ABA
responsiveness 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 alterations in guard cell ABA responses as observed for
Ga-deficient mutants. Therefore, we assessed both
ABA inhibition of light-induced stomatal opening
and the ABA promotion of stomatal closure in Ggdeficient plants. In agg1-1c, agg2-1, and agg2-2 single
mutants, stomatal responses to 50 mM ABA were not
statistically different from those of the wild type. A
wild-type ABA response was also observed in the
double agg1 agg2 mutant (Fig. 5, A and B). To ensure
that a subtle alteration in ABA sensitivity was not
overlooked in these experiments, the assays were
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Heterotrimeric G Proteins in Plants: Gg Subunits
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)
Blunt tip
Peduncle length (mm)
13.8 6 0.3
No
7.8 6 0.3
12.4 6 0.4*
Yes
13.0 6 0.5***
12.7 6 0.3*
Yes
13.2 6 0.3***
13.1 6 0.4
No
8.3 6 0.4
13.9 6 0.4
No
16.2 6 0.7***
14.1 6 0.5*
No
15.4 6 0.5***
repeated with a lower concentration of ABA (20 mM).
As shown in Figure 5, C and D, stomatal aperture
responses of the Gg-deficient single and double mutants at this lower ABA concentration still remained
indistinguishable from those of wild-type plants.
Many intracellular events contribute to the final
outcome of an alteration in stomatal aperture. One
well-defined aspect of the ABA inhibition of stomatal
opening is inhibition of the K1 channels that mediate
K1 uptake. To assess a more restricted ABA signaling
pathway in guard cells, we used the electrophysiological technique of patch clamping to evaluate the ABA
responsiveness of inward K1 currents. Ga- and Gbdeficient 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 in
Figure 6, ABA inhibited the inward K1 currents of
all of the Gg-deficient single and double mutants to the
same 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 outward
K1 channels that mediate K1 efflux during stomatal
closure was observed in wild-type Arabidopsis plants,
and the same absence of an ABA effect was observed
in Ga- and Gb-deficient mutants (Wang et al., 2001;
Coursol et al., 2003; L.M. Fan, unpublished data) as
well as in all of the Gg-deficient mutants (Fig. 6).
DISCUSSION
In the ‘‘canonical’’ model of heterotrimeric G protein
signal transduction, activity of the Gb subunit relies
heavily on its binding to the Gg subunit and subsequent membrane localization (Casey, 1995; Marrari
et al., 2007). In plants, as in animals and fungi, the Gb
subunit of the heterotrimeric G protein requires interaction with a Gg subunit for plasma membrane localization (Adjobo-Hermans et al., 2006; Zeng et al.,
2007). In fact, both canonical Arabidopsis Gg subunits
were shown to be prenylated and localized to the
plasma membrane (Adjobo-Hermans et al., 2006; Zeng
et al., 2007). Also similar to animals, plant Gb subunits
are tightly bound to Gg subunits, as shown in vitro
(Mason and Botella, 2000, 2001) and in vivo (Kato
et al., 2004; Adjobo-Hermans et al., 2006). Moreover,
overexpression of a mutated AGG1 lacking the isoprenylation motif resulted in a phenotype that resembles that of Gb-deficient mutants (Chakravorty and
Botella, 2007). These facts compel us to hypothesize
that in plants, as in animals, the bg subunits act as a
functional monomer. Therefore, Arabidopsis plants
lacking either or both of the two known Gg subunits
(AGG1 or AGG2) should display phenotypes that
totally or partially overlap those observed in mutants
lacking AGB1. Furthermore, a double AGG1 AGG2
knockout is expected to be identical to the AGB1deficient mutants in all respects. Indeed, this is the
case in many instances, and we previously reported
that lateral root formation, resistance to necrotrophic
pathogens, and germination on 6% Glc are similarly
altered in Gb- and Gg-deficient mutants (Trusov et al.,
2007). Additionally, the expression patterns of AGG1
and AGG2 resembled AGB1 expression in most plant
tissues (Trusov et al., 2007). On the other hand, the
unique ability of the plant Gg subunits to localize to
the plasma membrane independently of Gb (AdjoboHermans et al., 2006; Zeng et al., 2007; Wang et al.,
2008) raises the possibility that in Arabidopsis Gblacking mutants, free Gg subunits could be involved in
abnormal interactions. However, in many cases, this
remote possibility could be ruled out by comparing
Gb-deficient mutants with Ga-deficient mutants possessing Gbg dimers and not free Gg subunits.
Here, we show that there are considerable discrepancies between the behavior of Gb- and Gg-deficient
mutants, including the double agg1 agg2 mutant. AGB1
gene expression patterns in reproductive organs do
not match AGG1, AGG2, or their combination. Hypocotyl elongation, both in darkness and under light, as
well as hook development are altered in the Gbdeficient mutant but not in any of the single Ggdeficient mutants or in the double agg1 agg2 mutant.
Furthermore, agb1-2 was hypersensitive to ABA during germination, while all Gg-deficient mutants displayed wild-type sensitivity. Striking discrepancies
were observed in the morphological and developmental phenotypic characterization of the mutants. It is
remarkable that none of the aerial morphological traits
for which the Gb-deficient mutants show statistically
significant differences from the wild type can be
observed in the different Gg-deficient mutants. The
only instance in which Gg1-deficient mutants, and the
double agg1 agg2 mutant, show statistically significant
differences from the wild-type controls is in flowering
time; however, this effect was opposite to that observed in the Gb-deficient mutant (Table I). Finally, in
gpa1 (Wang et al., 2001; Coursol et al., 2003; Mishra
et al., 2006) and agb1 (L.M. Fan, unpublished data),
stomatal opening (but not stomatal closure) exhibits
hyposensitivity to inhibition by ABA. In contrast, no
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Trusov et al.
Figure 5. ABA regulates stomatal movements
similarly in Col and agg1-1c, agg2-1, agg2-2,
and agg1 agg2. A, ABA (50 mM) inhibition of
light-induced stomatal opening in Col, agg1-1c,
agg2-1, agg2-2, and agg1 agg2. Data shown are
means 6 SE from three replicates with n . 150
stomata for each experiment. B, ABA (50 mM)
induction of stomatal closure in Col, agg1-1c,
agg2-1, agg2-2, and agg1 agg2. Data shown are
means 6 SE from three replicates with n . 150
stomata for each experiment. C, ABA (20 mM)
inhibition of light-induced stomatal opening in
Col, agg1-1c, agg2-1, agg2-2, and agg1 agg2.
Data shown are means 6 SE from three replicates with n . 150 stomata for each experiment. D, ABA (20 mM) induction of stomatal
closure in Col, agg1-1c, agg2-1, agg2-2, and
agg1 agg2. Data shown are means 6 SE from
three replicates with n . 150 stomata for each
experiment.
ABA hyposensitivity of either ABA inhibition of stomatal opening or ABA promotion of stomatal closure
was observed in any of the single Gg-deficient mutants
or the double agg1 agg2 mutant. Stomatal aperture
responses result from a complex web of cellular signaling events that regulate multiple effectors (Li et al.,
2006). Therefore, we also assessed one particularly
well-defined subcellular event: ABA inhibition of inward K1 channels in guard cells (Blatt, 1990; Schwartz
et al., 1994). In Ga- and Gb-deficient lines, the response 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-deficient
mutants, guard cell K1 channel regulation by ABA was
present at the same magnitude as in the wild type.
Taken as a whole, our results demonstrate that the
absence of the Gg subunits does not completely phenocopy the lack of the Gb subunit.
To explain the observed discrepancies, two not
necessarily exclusive hypotheses could be proposed.
The first is that additional Gg subunits exist in
Arabidopsis, which could possibly be expressed in reproductive organs to explain the expression discrepancies observed between AGB1 and the two known Gg
subunit genes, AGG1 and AGG2. Although exhaustive
search of the fully sequenced Arabidopsis genome did
not reveal any additional canonical Gg subunits, these
subunits display poor sequence conservation; therefore, the presence of atypical subunits cannot be
discarded. This hypothesis allows retention of the
heterotrimeric G protein dogma, developed mainly
for mammalian systems, which states that all subunits
are interdependent and required for proper signaling
of the heterotrimer (Gilman, 1987).
A second hypothesis predicts some functional autonomy of the G protein subunits in plants, consistent
with several recent observations. In animals, interdependence of Ga subunits and the corresponding Gbg
dimers for correct subcellular localization has been
established (Takida and Wedegaertner, 2003), while in
plants, the subunits do not depend on each other
for plasma membrane targeting to the same extent
(Adjobo-Hermans et al., 2006; Zeng et al., 2007; Wang
et al., 2008). Moreover, both Arabidopsis Gg subunits
localized to the plasma membrane in Ga- and Gbdeficient mutants (Zeng et al., 2007). Other unusual
properties of the plant G protein complex recently led
Temple and Jones (2007) to suggest that the plant
heterotrimer is lagging behind in evolutionary terms
from its animal counterparts. This antiquity could
allow some functional autonomy for the individual
subunits, since it is logical to propose that the heterotrimer most probably originated from three initially
independent proteins. It is possible, therefore, that in
some processes, plant Ga and Gb subunits might act
independently from each other and from Gg subunits.
Notably, some Gg-dependent traits, such as susceptibility to necrotrophic fungi, methyl jasmonate sensitivity during root elongation and seed germination,
and lateral root number, were altered in an opposite
way in gpa1-4 compared with agb1-2 (Trusov et al.,
2006, 2007). At the same time, many traits reported
here, including ABA sensitivity in seed germination
and stomatal regulation, floral organ shape, and hy-
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Heterotrimeric G Proteins in Plants: Gg Subunits
Figure 6. ABA regulates K1 currents similarly in guard cells of Col, agg1-1c, agg2-1, agg2-2, and agg1 agg2. A, Typical whole
cell recordings of guard cell K1 currents with or without 50 mM ABA. Time and voltage scales shown in the top right panel apply
to all panels. B, Current/voltage relationship (mean 6 SE) of time-activated whole cell K1 currents as illustrated in A. Number of
guard 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).
pocotyl elongation in darkness, which were similarly
altered in gpa1-4 and agb1-2 mutants, were not altered
in Gg mutants. This could imply that Ga and Gb, but
not always Gg, are part of an as yet uncharacterized
complex, which governs those traits. Interestingly, in
rice, all individual heterotrimeric G protein subunits
were found as a part of 400-kD multiprotein complexes, but a fraction of the Gbg dimers were also
found not to be associated with those complexes (Kato
et al., 2004). In Arabidopsis, membrane-associated 400kD protein complexes were reported for the ERECTA
receptor-like kinase (Shpak et al., 2003), and knockouts
of ERECTA and ERECTA-like genes display similar
phenotypes to Ga- and/or Gb-deficient mutants in a
number of traits (Lease et al., 2001; Shpak et al., 2004)
as well as increased susceptibility to necrotrophic
pathogens (Llorente et al., 2005), suggesting that G proteins could be an integral part of those complexes.
Moreover, during the preparation of this article, it was
reported that in Arabidopsis roughly 30% of the native
Ga and all of the overexpressed cyan fluorescent
protein (CFP)-Gb are associated with large (approximately 700 kD) multiprotein complexes found in the
plasma membrane fraction (Wang et al., 2008). Unfortunately, no information on Gg-containing complexes
was provided for either of the Gg subunits (Wang
et al., 2008).
The question arises whether plant Gb can act independently of Gg, as has already been described for
Gb5 in animal systems. The mammalian Gb5 subunit
can form functional dimers with a number of RGS
proteins that contain a ‘‘Gg-like motif’’ known as the
GGL domain, precluding formation of the canonical
dimer Gb5g (Snow et al., 1998; Witherow et al., 2000;
Sondek and Siderovski, 2001; Witherow and Slepak,
2003; Willars, 2006). There are no known GGL domaincontaining plant proteins; reports about plant GGL
domain proteins, however, are currently available. In
addition, the WD40 propeller structure of the Gb
subunit allows interaction with multiple proteins,
hence providing a scaffold for large protein assemblies. In Arabidopsis and tobacco (Nicotiana tabacum),
Plant Physiol. Vol. 147, 2008
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Trusov et al.
the Gb subunit was identified in plasma membrane
Triton X-100-insoluble microdomains along with other
signaling components, including kinases and small
GTP-binding proteins (Peskan and Oelmuller, 2000;
Peskan et al., 2000; Shahollari et al., 2004). Thus, it is
not unlikely that Gb could function independently
from Gg as an integral part of multiprotein complexes
to mediate some processes while functioning as a Gbg
dimer in others. In this scenario, knockout of AGB1
would destroy both the Gbg dimers and the hypothetical Gb-containing complexes, while the absence
of Gg subunits would only obliterate signaling that
was directly dependent on Gbg dimers.
In both hypotheses, it is necessary to explain how
Gb can be targeted to the plasma membrane in the
absence of both Gg subunits. A hybrid CFP-AGB1
fusion protein showed diffuse localization in the cytoplasm unless coexpressed with either AGG1 or
AGG2 subunits (Adjobo-Hermans et al., 2006). This
study demonstrated sufficiency of the Gg subunits for
the plasma membrane localization of Gb. However,
since this study was based on high ectopic expression
of the AGB1, AGG1, and AGG2 genes, it is possible that
other proteins, or complexes, can also anchor Gb to the
plasma membrane under normal circumstances. In
this respect, it will be interesting to determine whether
AGB1 is membrane localized in the agg1 agg2 double
mutant. Just as for all other known Gg subunits, the
ability of AGG1 and AGG2 to be targeted to the
plasma membrane crucially relies on prenylation of
the C-terminal CAAX motif (Zeng et al., 2007). In
Arabidopsis, two prenylation enzymes, geranylgeranyltransferase I (PGGT-I) and farnesyltransferase (PFT),
have been identified (Caldelari et al., 2001; Running
et al., 2004). Interestingly, knockout of PFT results in
ABA hypersensitivity and phenotype alterations similar to those observed in Ga- and Gb-deficient mutants, while knockout of PGGT-I, which has been
shown to prenylate both AGG1 and AGG2 subunits
(Zeng et al., 2007), leads to wild-type phenotype and
wild-type ABA sensitivity in seed germination (Johnson
et al., 2005; Zeng et al., 2007). Taken together with
the fact that knockout of both Gg subunits does not
completely phenocopy Gb-deficient mutant phenotypes, these observations suggest that there are additional, probably farnesylated, proteins interacting with
AGB1 in Arabidopsis. It is possible, therefore, that
currently unknown Gg subunits, GGL-containing
RGSs, or some components of the multiprotein complexes described by Wang et al. (2008) can be prenylated by PFT and are able to target AGB1 to the
plasma membrane. It is interesting that in rice, the Gg
subunit RGG2 lacks the C-terminal prenylation motif
but nevertheless is detected in the plasma membrane
fraction associated with Gb (Kato et al., 2004).
CONCLUSION
Additional studies will prove or disprove the feasibility of the hypotheses described above. Neverthe-
less, whatever the result of those studies, our data
unambiguously reveal that the variety of heterotrimeric G proteins in plants is not limited to the two
canonical heterotrimers Gabg1 and Gabg2 found so
far. This adds an additional level of complexity to the
molecular mechanisms used by G proteins in plants
and provides a new degree of functional selectivity to
that reported previously for the two known heterotrimers (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 developmental 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 (PhytoTechnology 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
646
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Heterotrimeric G Proteins in Plants: Gg Subunits
plates for analysis (Molecular Dynamics). For RT-PCR, reverse transcriptions
were carried out using the SuperScript III RT kit according to the manufacturer’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 described previously (Trusov et al., 2007).
Stomatal Aperture Experiments and Guard
Cell 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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