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Plant Cell. Apr 2007; 19(4): 1235–1250.
PMCID: PMC1913745

Heterotrimeric G Protein γ Subunits Provide Functional Selectivity in Gβγ Dimer Signaling in Arabidopsis[OA]

Abstract

The Arabidopsis thaliana heterotrimeric G protein complex is encoded by single canonical Gα and Gβ subunit genes and two Gγ subunit genes (AGG1 and AGG2), raising the possibility that the two potential G protein complexes mediate different cellular processes. Mutants with reduced expression of one or both Gγ genes revealed specialized roles for each Gγ subunit. AGG1-deficient mutants, but not AGG2-deficient mutants, showed impaired resistance against necrotrophic pathogens, reduced induction of the plant defensin gene PDF1.2, and decreased sensitivity to methyl jasmonate. By contrast, both AGG1- and AGG2-deficient mutants were hypersensitive to auxin-mediated induction of lateral roots, suggesting that Gβγ1 and Gβγ2 synergistically inhibit auxin-dependent lateral root initiation. However, the involvement of each Gγ subunit in this root response differs, with Gβγ1 acting within the central cylinder, attenuating acropetally transported auxin signaling, while Gβγ2 affects the action of basipetal auxin and graviresponsiveness within the epidermis and/or cortex. This selectivity also operates in the hypocotyl. Selectivity in Gβγ signaling was also found in other known AGB1-mediated pathways. agg1 mutants were hypersensitive to glucose and the osmotic agent mannitol during seed germination, while agg2 mutants were only affected by glucose. We show that both Gγ subunits form functional Gβγ dimers and that each provides functional selectivity to the plant heterotrimeric G proteins, revealing a mechanism underlying the complexity of G protein–mediated signaling in plants.

INTRODUCTION

Heterotrimeric G proteins are an important element of transmembrane signal transduction, coupling stimuli as diverse as light, neurotransmitters, odorants, tastants, and hormones. They are found in a variety of eukaryotic organisms, including plants, fungi, and animals. The classical heterotrimer consist of three different subunits, α, β, and γ, which are organized in a highly conserved structure and typically bound to specific G protein–coupled receptors. Activation of the receptor by ligand binding induces a conformational change in Gα, catalyzing the exchange of GDP to GTP. GTP loading causes a protein conformational change that promotes dissociation of the heterotrimer into two functional signaling elements: the Gα subunit and the Gβγ dimer. These two elements (functional subunits) interact with specific effector molecules controlling downstream signaling. The inherent GTPase activity of the Gα subunit hydrolyzes its bound GTP, leading to the reassociation of Gα and the Gβγ dimer, returning the heterotrimer to its inactive GDP-bound state. While interaction between Gα and the Gβγ dimer is dependent on the conformational status of the Gα subunit, interaction between Gβ and Gγ is essentially nondissociable; therefore, the Gβγ dimer acts as a single functional unit in the cell (Gautam et al., 1998).

It was initially thought that signaling in animals only occurred via the activated Gα subunit, with the role of Gβγ being to inhibit the action of Gα by reforming the inactive heterotrimer and guiding Gα back to the receptor for reactivation. However, it is now established that the Gβγ dimer is an active signaling factor in at least as many processes as the Gα subunit (Clapham and Neer, 1997). Among others, the Gβγ dimer is able to interact with adenylyl cyclases, potassium channels, and phospholipases (Clapham and Neer, 1993; Scott et al., 2001). Aside from the activation of specific downstream effectors, the Gβγ dimer is involved in receptor recognition (Lim et al., 2001), membrane targeting, and activation of the Gα subunit (Evanko et al., 2000, 2001). Binding between Gα and Gβγ occurs at a molecular interface largely contained within the β-propeller structure of Gβ. With the exception of Gβ5, there is little binding preference between Gα and Gβ pairs. Therefore, it is assumed that Gγ provides a major share of the structural requisite for the selective coupling of the heterotrimer to the receptor and the Gβγ dimer to its effectors (Gautam et al., 1990; Simon et al., 1991; Hou et al., 2000; Myung and Garrison, 2000; Azpiazu and Gautam, 2002; Chen et al., 2005; Myung et al., 2006). Recent evidence indicates that some animal Gβγ dimers can move from the plasma membrane to the Golgi upon receptor activation, providing an extra element of spatial segregation to the Gβγ dimer in G protein–mediated signaling. The Gγ subunit type and the Gα subunit nucleotide exchange properties strongly influence the rate of translocation (Akgoz et al., 2004, 2006; Azpiazu et al., 2006).

A characteristic of mammalian systems is the existence of gene families for each of the G protein subunits. At least 23 Gα subunits, 6 Gβ subunits (including an alternatively spliced variant), and 12 Gγ subunits (Gautam et al., 1998; Balcueva et al., 2000) have been reported in humans, but not all possible combinations are present in the cell, with combinatorial multiplicity of Gβγ dimers being restricted by the specific expression patterns of the genes and selective interactions between different Gβ and Gγ subunits. Nevertheless, a wide range of Gβγ dimers, serving as distinct signal transduction elements involved in different processes, have been described (Camps et al., 1992; Katz et al., 1992; Chen et al., 1997; Clapham and Neer, 1997; Gautam et al., 1998; Bommakanti et al., 2000; Mirshahi et al., 2002; Krystofova and Borkovich, 2005).

In contrast with mammalian systems, only one canonical Gα subunit gene (GPA1) (Ma et al., 1990), one canonical Gβ subunit gene (AGB1) (Weiss et al., 1994), and two Gγ subunit genes (AGG1 and AGG2) (Mason and Botella, 2000, 2001) have been found in the Arabidopsis thaliana genome. The same number of G protein subunits were reported in the monocot species rice (Oryza sativa) (Ishikawa et al., 1995, 1996; Iwasaki et al., 1997; Kato et al., 2004); however, two Gα subunits were described for legume species (Kim et al., 1995; Gotor et al., 1996; Marsh and Kaufman, 1999). G proteins are implicated in a large variety of processes in plants (Jones and Assmann, 2004; Perfus-Barbeoch et al., 2004; Assmann, 2005; McCudden et al., 2005; Temple and Jones, 2007); nevertheless, specific signaling roles for the Gα subunit or Gβγ dimers remained elusive until recently. Analysis of T-DNA and ethyl methanesulfonate mutants lacking functional Gα or Gβ subunits showed that both Gα and Gβγ could be involved in specific and independent pathways (Ullah et al., 2003; Joo et al., 2005; Chen et al., 2006a; Pandey et al., 2006; Trusov et al., 2006) as well as in the same processes (Ullah et al., 2003; Pandey et al., 2006). Studies using Arabidopsis demonstrated that the Gβ-deficient agb1-1 and agb1-2 mutants have flowers with elongated peduncles, shortened flat-top siliques, rounded rosette leaves with crinkled surfaces, and increased root mass (Lease et al., 2001; Ullah et al., 2003). Detailed studies revealed that Gβ modulates lateral root formation by interfering with auxin-dependent cell division (Ullah et al., 2003). It was shown that Gβ-mediated signaling, but not Gα, plays a distinct part in plant resistance against necrotrophic pathogens (Llorente et al., 2005; Trusov et al., 2006). Specific changes in seed germination were also ascribed to Gβ activity (Pandey et al., 2006; Trusov et al., 2006). Finally, analysis of transgenic tobacco (Nicotiana tabacum) plants with reduced Gβ subunit levels due to antisense expression of the Gβ subunit mRNA suggested that the Gβ subunit is involved in regulation of the reproductive phase of the tobacco life cycle, particularly in stamen development and pollen maturation (Peskan-Berghofer et al., 2005).

Strong interaction between plant Gβ and each Gγ subunit was demonstrated in vitro (Mason and Botella, 2000, 2001) as well as in vivo (Kato et al., 2004; Adjobo-Hermans et al., 2006, Chakravorty and Botella, 2007). However, despite sequence similarity (48% amino acid identity), the interaction between each of the two Arabidopsis Gγ subunits and Gβ seems to be centered in different domains of the protein (Mason and Botella, 2000, 2001; Temple and Jones, 2007).

Nothing is known about the cellular and physiological roles of either of the two known Gγ subunits, their possible functional redundancy, and whether the two potential dimers, Gβγ1 and Gβγ2, are involved in the same or different signaling pathways. We took advantage of the extensive phenotypic characterization of loss-of-function agb1 mutants, and using this inventory of phenotypes, we asked which of the Gγ subunits acts with Gβ to regulate a specific function. Fungal resistance, root development, and glucose sensing were the three well-characterized AGB1-signaling pathways examined in this study. By a genetic approach, we dissected the roles of the Gγ subunits in G protein signaling in these pathways. Our results show that the different Gγ subunits form independent signal-transducing Gβγ dimers and impart functional selectivity to the heterotrimeric G protein signaling network.

RESULTS

The Expression Profiles of AGG1 and AGG2 Are Distinct but Together Overlap AGB1 Expression

Expression patterns for Gα and Gβ subunit genes were previously reported in various plant species (Weiss et al., 1993; Huang et al., 1994; Kaydamov et al., 2000; Perroud et al., 2000; Chen et al., 2006c). In order to study the tissue-specific and developmental regulation of the AGB1, AGG1, and AGG2 genes, transgenic Arabidopsis (Col-0) plants were produced containing the promoter regions of each gene fused to the β-glucuronidase (GUS) reporter gene. At least three independent lines were characterized for each of the promoter constructs. Transgenic plants did not show any obvious morphological alterations, suggesting that inserts did not disrupt functional genes. GUS histochemical assays revealed that all three genes are active during early seedling development, with GUS activity detected throughout the plant but highest at the hypocotyl–root junction in 2-d-old AGB1:GUS seedlings (Figure 1A). AGG1:GUS staining was observed in the hypocotyl, while AGG2:GUS staining occurred in the upper part of the root, including root hairs, and gradually declined along the root (Figure 1A).

Figure 1.
In Situ AGB1, AGG1, and AGG2 Expression Patterns.

During later development, all three genes always showed cell/tissue-specific expression patterns, although the overall intensity of the stain was always higher in soil-grown versus plate-grown plants. In rosette leaves of AGB1:GUS plants, intense GUS staining was detected in veins and guard cells (Figures 1B and 1C). AGG1 expression was restricted to veins, while AGG2 expression was observed primarily in guard cells (Figure 1C). Interestingly, all three genes were found to be expressed in hydathods, specialized leaf organs responsible for the excretion of excessive water and/or salts, but while AGG2 always showed strong staining, AGB1 and AGG1 only occasionally did so (Figure 1B; see also Figure 3A below).

Figure 3.
The Gγ Subunit Is Involved in Defense against Necrotrophic Fungi.

In roots, AGG1 expression was restricted to the stele (Figures 1D and 1E). By contrast, AGG2 expression was, with one exception, excluded from the stele yet found in the cortex and epidermis (Figure 1E). Neither AGG1 nor AGG2 expression was homogeneous in its respective tissues along the root length. The exception to the exclusion of AGG2 expression in the stele was found in young plants (5 to 7 d old) grown on Murashige and Skoog (MS) medium, in which weak AGG2 expression was observed in the central cylinder and not in outer tissues. AGB1 is expressed in all root cell types (Figures 1D and 1E) (Chen et al., 2006c). Three distinct expression patterns were observed in AGB1:GUS plants: only in the stele, the cortex, or the entire section, with the least intensity or no staining in endodermis/pericycle cells (Figure 1E). It is interesting that throughout the plant, AGG1 and AGG2 expression patterns rarely overlapped and together matched the expression of AGB1 in most tissues (with the exception of flowers and siliques).

Loss-of-Function Mutants for the Gγ Subunits

In order to study the function of both Gγ subunits in Arabidopsis, mutants carrying T-DNA insertions in AGG1 (agg1-1w, on the Wassilewskija [Ws] ecotype) and AGG2 (agg2-1, on the Columbia-0 [Col-0] background) genes were identified. An AGG1-deficient mutant in the Col-0 background was generated by genetic introgression over eight successive generations, resulting in a line designated agg1-1c (backcross to Col-0). In agg1-1w, the T-DNA insertion is positioned within the second intron, splitting the protein in approximately two equal halves, while in agg2-1, two tandem and opposing T-DNA insertions are located in the third intron, disrupting the C-terminal region of the hypothetical protein (Figure 2A). RT-PCR analysis showed that neither allele (agg1-1c or agg2-1) produces a detectable functional transcript for its respective gene (Figure 2C). In addition, the absence of AGG1 expression in the agg1-1c mutants did not result in any observable changes in AGG2 expression, due to possible compensatory effects (Figure 2C; data not shown). The reverse applies to agg2-1 mutants. A double knockout of the AGG1 and AGG2 genes was obtained by hybridization of the agg1-1c and agg2-1 mutants (agg1 agg2). As expected, this line lacked detectable expression of each of the two Gγ subunit genes (Figure 2C).

Figure 2.
Molecular Characterization of agg1-1, agg2-1, and RNAi Mutants.

In addition to the T-DNA mutants, transgenic lines containing RNA interference (RNAi) constructs designed to individually silence either AGG1 or AGG2 (agg1RNAi and agg2RNAi, respectively) were produced in Col-0 (Figure 2B). After screening a large number of individual transgenic lines for each targeted gene, a single-insertion, homozygous line with no detectable expression was selected for further analysis (Figure 2C). For the sake of clarity, agg1-1c and agg1RNAi lines will be collectively referred to as agg1 mutants in the text, while agg2-1 and agg2RNAi lines will be collectively named agg2 mutants.

Gβγ-Mediated Defense against Necrotrophic Fungi Is Selectively Mediated by AGG1 but Not AGG2

It was shown previously that Gβγ-mediated signaling, but not Gα-mediated signaling, is involved in resistance against necrotrophic fungi (Llorente et al., 2005; Trusov et al., 2006). Therefore, we sought to determine whether there is a specific Gγ subunit engaged with Gβ in this process or whether both subunits play redundant or synergistic roles. In preliminary experiments, we analyzed the behavior of all three genes (AGB1, AGG1, and AGG2) in response to attack by necrotrophic pathogens using transgenic plants carrying the promoter:GUS fusion constructs. Alternaria brassicicola is an air-borne avirulent pathogenic fungus of Arabidopsis ecotype Columbia (Penninckx et al., 1996; Schenk et al., 2000, 2003; Thomma et al., 2000; van Wees et al., 2003), even though some isolates can reproduce at a very low rate under favorable conditions (van Wees et al., 2003). When plants were inoculated with a suspension of A. brassicicola spores, elevated GUS activity was detected 24 h after infection in AGB1:GUS and AGG1:GUS but not in AGG2:GUS transgenic plants (Figure 3A). GUS staining was restricted to the inoculation site and did not spread throughout the entire leaf.

Fusarium oxysporum (f. sp conglutinans) is a soil-borne necrotrophic fungus that uses the root tip, secondary root formation foci, and wounds as entry points. It subsequently colonizes the plant by traveling through the vascular system (Mauchmani and Slusarenko, 1994; Agrios, 2005). In contrast with A. brassicicola, F. oxysporum is a virulent pathogen of Arabidopsis (Berrocal-Lobo and Molina, 2004). Surprisingly, inoculation of roots with F. oxysporum did not induce GUS activity in root tissue above background levels in any of the three reporter lines; however, significant induction was detected in leaves of AGB1:GUS and AGG1:GUS plants (Figure 3B). No induction was observed in AGG2:GUS plants; rather, a slight decrease in gene expression was observed in leaves and roots. Taken together, our findings indicate that leaf expression of AGB1 and AGG1 is systemically activated by F. oxysporum and locally by A. brassicicola.

To understand the roles of Gγ1 and Gγ2 in resistance against necrotrophic pathogens, we assayed the response of the T-DNA mutants agb1-2, agg1-1c, agg2-1, agg1 agg2, and the RNAi lines (agg1RNAi and agg2RNAi) to A. brassicicola and F. oxysporum inoculation. Roots of 2-week-old mutant and wild-type plants were infected with bud cell suspensions of F. oxysporum, and disease progression was monitored over time from the development of the first symptoms until plants died. Figure 3C illustrates the appearance of typical disease symptoms at an early stage of infection. The advanced chlorosis observed in veins and leaves of agb1-2, agg1-1c, agg1RNAi, and agg1 agg2 mutants gives a qualitative indication that there is increased susceptibility to F. oxysporum in these lines compared with the wild type as well as agg2-1 and agg2RNAi mutants. To quantify the levels of resistance, the number of decayed plants in all mutant lines and wild-type controls was determined (Figure 4A). Plants lacking green leaves were considered decayed. agg1-1c, agg1RNAi, and agg1 agg2 lines showed similar dynamics to agb1-2, all of them exhibiting a faster rate of disease progression than wild-type plants, while the behavior of agg2-1 and agg2RNAi mutants resembled that of the wild type. To test whether the loss of AGG1 had a similar effect in the Ws background, we compared the agg1-1w mutant (in Ws) with wild-type Ws and the Gα subunit null mutant gpa1-1 (also in the Ws ecotype) (Ullah et al., 2001). Unfortunately, no agb1 mutants are yet available in the Ws background. We previously showed that the Gα subunit null mutants gpa1-3 and gpa1-4 (Col-0 ecotype) have slightly enhanced resistance to F. oxysporum (Trusov et al., 2006). After performing inoculation and disease evaluation as for Col-0 lines, it was evident that disease progressed faster in agg1-1w plants than in the Ws wild type, while gpa1-1, as expected, displayed slightly enhanced resistance (Figure 4C). The differences in disease progression observed between agg1, agb1, and agg1 agg2 mutants compared with wild-type Col-0 and agg2 mutants were statistically significant (P < 0.05). Similarly, the differences observed between agg1-1w and the wild type and between gpa1-1 and the wild type in the Ws ecotype were statistically significant (P < 0.01). All experiments were repeated at least twice with similar results.

Figure 4.
Differential Responses of Gγ-Deficient Mutants to Pathogen Attack and MeJA Treatment.

Vegetative growth was also impaired, albeit to different degrees, in wild-type and mutant plants infected with F. oxysporum. Figure 4B shows the inhibition of rosette growth expressed as relative size (rosette diameter) of Fusarium-inoculated versus mock-inoculated plants of the same genotype. The growth of both agg1 mutants, the agg1 agg2 double mutant, and agb1-2 was significantly affected by the pathogen at 5 d after inoculation (P < 0.05), while agg2 mutants and wild-type plants were almost indistinguishable from their respective mock-inoculated controls. By day 15, the rosette diameter of Fusarium-infected wild-type and agg2 mutants was almost half that of their mock-inoculated controls, while the agg1 mutants, the agg1 agg2 double mutant, and agb1-2 were more severely affected. Absolute values (day 15) for the mean rosette diameter of mock-inoculated wild-type (Col-0), agb1-2, agg1-1c, agg1RNAi, agg2-1, agg2RNAi, and agg1 agg2 plants were 55.2 ± 6.7, 41.1 ± 6.1, 53.9 ± 8.1, 54.3 ± 6.0, 57.5 ± 9.2, 58.1 ± 11.5, and 49.9 ± 9.3 mm, respectively (shown as averages ± se), while leaves inoculated with F. oxysporum displayed measurements of 34.8 ± 5.1, 11.3 ± 3.5, 18.7 ± 4.4, 17.3 ± 5.6, 30.6 ± 8.4, 33.2 ± 8.1, and 15.0 ± 4.6 mm, respectively.

We previously showed that Gβ is also involved in resistance to A. brassicicola (Trusov et al., 2006). Application of spores (106 spores/mL) on the leaf surface of Arabidopsis plants causes necrotic lesions that are clearly different in the wild type and Gβ-deficient mutants. agb1-2, agg1, agg2, and agg1 agg2 mutants along with wild-type Col-0 plants were inoculated with A. brassicicola (Figure 3D), and disease progression was quantified by measuring the necrotic lesion area (given as a percentage of the droplet-inoculated area) (Figure 4E). Statistical analysis showed two very distinct groups that are significantly different from each other (P < 0.05). Lesions on agb1-2, agg1, and agg1 agg2 mutant leaves occupied ~50 to 60% of the inoculated area, in contrast with wild-type plants and agg2 mutants, in which an average of 30% of the inoculated area became necrotic. In agreement with these observations, RNA gel blot hybridization revealed that 20 h after infection with A. brassicicola, steady state levels of the plant defensin PDF1.2 transcript were reduced in agb1-2, agg1, and agg1 agg2 mutants compared with the wild type and agg2 mutants (Figure 4D).

It was previously established that the increased susceptibility to fungal necrotrophic pathogens that was observed in Gβ-deficient mutants correlates with a decreased sensitivity to methyl jasmonate (MeJA). Therefore, we assayed MeJA sensitivity using a germination assay. All mutants showed reduced sensitivity to MeJA compared with wild-type plants (Figure 4F), although to different degrees: agb1-2 = agg1 agg2 < agg1 < agg2 < wild type. MeJA sensitivity was also assayed using root length inhibition assays (Figure 4G). Two statistically different groups (P < 0.05) were observed, the first one showing decreased sensitivity to MeJA in agb1-2, agg1 agg2, and agg1 mutants and the second one containing the wild type and agg2 mutants.

AGG1 and AGG2 Act Additively in Gβγ-Mediated Lateral Root Development

It has been established that Gβ, but not Gα, attenuates auxin-induced cell division leading to lateral root proliferation, although it does not directly couple auxin signaling (Ullah et al., 2003; Chen et al., 2006a). Figure 5A shows the number of lateral roots in 2-week-old wild-type plants and mutants deficient in Gβ, Gγ1, Gγ2, or both Gγ subunits grown on vertical plates (0.5× MS, 1% sucrose, and 0.8% agar, 16:8 day:night cycle, 23°C). All mutants produced more lateral roots than wild-type plants, but three statistically distinct groups (P < 0.05) were observed within the mutants: agb1-2 and double agg1 agg2 mutants had the highest number of lateral roots, agg2-1 and agg2RNAi mutants produced fewer lateral roots, while agg1-1c and agg1RNAi had even fewer roots (Figure 5A). Alteration of the growth conditions, such as an increase in MS salt concentration (from 0.5× to 1×) and reduced temperature (from 23 to 21°C) substantially (more than three times) decreased the total number of lateral roots (Figure 5C, white bars) as well as the differences among the various mutants and between mutants and the wild type.

Figure 5.
Effect of the Loss of Gγ Subunits on Lateral Root Formation.

To assay responsiveness to exogenous auxin, seedlings were grown on medium supplemented with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) and then transferred to growth medium (1× MS) in the presence or absence of 1-naphthaleneacetic acid (NAA) for 5 d before scoring the number of lateral roots (Figure 5B) (Himanen et al., 2002; Ullah et al., 2003). All of the tested G protein mutants showed increased sensitivity to NAA compared with wild-type plants. The ratio of lateral roots developed on NAA-containing medium versus control medium gives an additional indication of the relative sensitivity to NAA: Col-0, 2.2; agb1-2, 3.9; agg1-1c, 4.1; agg1RNAi, 3.6; agg2-1, 3.4; agg2RNAi, 3.6; and agg1 agg2, 4.0.

Exposure of Arabidopsis plants to high temperature (29°C) results in an increase in endogenous auxin levels (Gray et al., 1998). Although that original work focused on the effect of endogenous auxin induction on hypocotyl elongation, an increased number of lateral roots was also observed (Gray et al., 1998). In addition, it has been established that shoot-derived auxin is required for the emergence of lateral root primordia (Reed et al., 1998). agb1-2, agg1, agg2, and agg1 agg2 mutants along with wild-type Col-0 plants were grown at either 21 or 29°C (1× MS), and the number of lateral roots was determined in 2-week-old plants. All genotypes showed a marked increase in the number of lateral roots when grown at high temperature, with the smallest effect (~2.5-fold increase) observed in wild-type plants (Figure 5C). agg1-1c and agg1RNAi mutants displayed 5.5- and 4.6-fold increases, respectively, while agg2-1 and agg2RNAi showed 3.2- and 3.5-fold increases, respectively. Both agb1-2 and double agg1-1 agg2-1 mutants produced approximately seven times more lateral roots when grown at 29°C (Figure 5C). In addition, adventitious roots were frequently observed (80 to 90% of seedlings) on hypocotyls of agb1-2, agg1 agg2, and agg1 mutants but never in wild-type plants or agg2 mutants (data not shown).

AGG1 and AGG2 Are Involved in the Modulation of Acropetally and Basipetally Transported Auxin Activity, Respectively

AGG1 and AGG2 expression in roots is cell-specific (Figure 1D), correlating with acropetal and basipetal auxin streams, respectively (Mitchell and Davies, 1975; Jones, 1998). Therefore, we hypothesized that Gβγ1 represses lateral root development from the central cylinder by attenuating the activity of acropetally transported auxin, while Gβγ2 represses lateral root formation or growth through the cortex/epidermis by affecting basipetal auxin. It was established that shoot-derived auxin is the predominant source of auxin in young (5- to 7-d-old) Arabidopsis roots, controlling lateral root emergence during early development, while later in development, the root system gradually reduces the dependence on shoot-derived auxin by synthesizing a sufficient amount within the root tip at 10 d after germination (although shoot-derived auxin is still important for primordial outgrowth) (Bhalerao et al., 2002; Ljung et al., 2005). Therefore, seedlings were grown for 7 d (1× MS) to allow maximal root elongation before the root tip started to produce auxin, and then acropetal auxin transport was inhibited by the method described by Reed et al. (1998). Seedlings with the auxin transport inhibitor NPA block placed at the root tip had only acropetal auxin transport in the area of the root above the block, while seedlings with the NPA block placed at the shoot–root junction should develop lateral roots mainly under the control of basipetal transport, with the exception of the fraction of roots initiated by early acropetal auxin. The dynamics of lateral root emergence was recorded during the 2-week period after the application of the NPA block (Figures 6A and 6B). As expected, the rate of lateral root production after both treatments was highest in the agb1-2 and agg1 agg2 mutants and lowest in wild-type plants. agg1-1c seedlings produced abundant lateral roots (statistically indistinguishable from agb1-2 and agg1 agg2), despite the arrest of basipetal transport (Figure 6A). Inhibition of acropetal transport resulted in an initially high number of lateral roots in agg1-1c seedlings (day 13 in Figure 6B), probably as a result of early acropetal auxin flux before the block was applied. After the initial peak, the rate of lateral root formation was similar to that in wild-type plants (Figure 6B). By contrast, suppression of basipetal transport reduced lateral root numbers in agg2-1 to wild-type levels (Figure 6A), while arrest of acropetal transport resulted in elevated levels of lateral roots, statistically indistinguishable from those of agb1-2 and agg1 agg2 mutants (Figure 6B). Similar behavior was exhibited by the RNAi lines (data not shown).

Figure 6.
Specific Roles of AGG1 and AGG2 in the Regulation of Auxin Response.

To provide further evidence for the selective roles of the Gγ1 and Gγ2 subunits in roots, we analyzed two specific processes dependent upon the two different auxin streams, adventitious root formation in hypocotyls and root gravitropism. Adventitious root formation predominantly relies on auxin transported within the hypocotyl stele (Liu and Reid, 1992; Nicolas et al., 2004). Aseptically excised wild-type and mutant hypocotyls were incubated with the synthetic auxin NAA. agb1-2, agg1-1c, and agg1 agg2 mutants formed adventitious roots throughout the entire hypocotyl, while in wild-type plants and the agg2-1 mutant adventitious roots were not formed or were present only near the ends of the hypocotyl segments (Figure 6C).

Rashotte and coworkers (2000) showed that inhibition of basipetal auxin transport in roots completely blocked its gravity response, while inhibition of acropetal transport only partially reduced it. Therefore, we assayed the gravitropic response of wild-type and G protein mutant roots by measuring the root angle (measured from the horizontal position) at 24 h after gravistimulation. Figure 6D shows that agb1-2, agg2-1, and agg1 agg2 mutants were less responsive to gravistimulation than wild-type plants and agg1-1c (P < 0.001). Interestingly, agg1-1c was slightly less responsive than the wild type (P < 0.05), probably due to a limited participation of the acropetal auxin in the gravity response (Rashotte et al., 2000).

AGG1 and AGG2 Are Involved in Different Responses during Germination

Two recent reports established that Gβ signaling plays a role in germination (Pandey et al., 2006; Trusov et al., 2006). To determine the specific roles of each of the partner Gγ subunits in this process, mutants lacking Gβ, Gγ1, Gγ2, or both Gγ subunits were subjected to germination tests. Since germination efficiency is extremely sensitive to the growth conditions experienced by the parental plant and postharvest storage, all seed lots were collected at the same time from plants grown simultaneously under the same conditions and were stored for 2 months at 4°C in the dark. Approximately 100 sterilized seeds of all tested lines were planted on the same Petri dish for a single treatment.

Germination and early development are regulated by many Gβγ-mediated signals, and glucose is arguably the best characterized of those signals to date (Ullah et al., 2002; Pandey et al., 2006; Wang et al., 2006). As shown in Figure 7A, there was a clear difference between wild-type and mutant plants when germinated in the presence of 6% glucose, while 4% glucose did not discriminate among the different genotypes and 2% glucose resulted in nearly 100% germination. Because light intensity also has an effect on germination, we used two different intensities of continuous light irradiation (63 and 150 μmol·m−2·s−1). The higher light intensity resulted in faster germination rates, reaching 90% by day 6 on glucose and by day 3 on mannitol (Figures 7C and 7E, respectively), obscuring any differences between genotypes. By contrast, the slower germination rates observed using a lower light intensity accentuated the differences among genotypes. When sown on glucose under low light intensity, agb1-2, agg1, and agg1 agg2 mutant seeds showed drastically reduced germination rates compared with wild-type seeds, with <50% germination after 2 weeks (Figure 7B). By contrast, at higher light intensities, the differences between wild-type and agb1 and agg1 mutant seeds were only observed at day 2 (Figure 7C). Interestingly, agg2 mutants also displayed significant inhibition of germination on glucose, albeit at notably lower levels than agg1 mutants. Again, the difference was statistically significant in lower light (Figure 7B), while at higher light this difference was insignificant (Figure 7C).

Figure 7.
Germination Assays in Gγ-Deficient Mutants.

To discriminate between the signaling effect and the osmotic stress component observed when plants are exposed to high levels of sugar, we determined the effect of the osmotic agent mannitol on germination at two light intensities. Surprisingly, mannitol severely decreased germination rates in agb1-2, agg1, and agg1 agg2 mutants at all time points under the lower light intensity (Figure 7D) and at day 2 under higher light (Figure 7E). By contrast, agg2 mutants initially showed low germination rates but quickly reached wild-type levels by day 6 under low light (Figure 7D) and were indistinguishable from the wild type under higher light intensity at all time points (Figure 7E).

DISCUSSION

Previously, the functional selectivity of Gγ subunits was largely unrecognized, with the general view that Gγ function is limited to anchoring the Gβγ dimer to the membrane. However, Gγ recently emerged as an important element that provides effector specificity as well as receptor selectivity for the heterotrimer (Gautam et al., 1990; Hou et al., 2000; Akgoz et al., 2002; Azpiazu and Gautam, 2002; Myung et al., 2006).

The initial discovery of single Gα and Gβ subunits in Arabidopsis challenged the concept that plants use combinatorial subunit composition to define G protein receptor/effector specificity (Arabidopsis Genome Initiative, 2000), as proven in mammalian systems (Robishaw and Berlot, 2004). With the recent discovery of two Gγ subunits in Arabidopsis (Mason and Botella, 2000, 2001), we must now address this possibility. Since both plant Gγ subunits share a number of similarities with animal Gγ subunits, such as the strong interaction with Gβ and the presence of isoprenylation domains, it is reasonable to expect that there are two operational Gβγ subunits in Arabidopsis. A number of logical questions follow, such as whether the two subunits mediate the same processes or whether they specialize in different developmental, biotic, or abiotic responses. In this respect, it is interesting that AGG1 and AGG2 in situ expression profiles show a high degree of tissue specificity and that, even though the sum of their individual expression patterns mimics the overall Gβ expression, the two Gγ gene expression patterns rarely overlap. This raises the possibility that Gγ subunits impose selective functionality restricted by expression patterns.

The functions of the two Gγ subunits are intrinsically linked to Gβ, since, based on mammalian studies, the Gβγ dimer operates as a single signaling unit. The Gβ subunit has been associated with a number of processes using loss-of-function mutants (Lease et al., 2001; Ullah et al., 2003; Llorente et al., 2005; Pandey et al., 2006; Trusov et al., 2006). However, according to the classical mechanism of heterotrimeric G protein action, the lack of a functional Gβ subunit affects not only processes directly mediated by Gβ but also those mediated by Gα; therefore, some of the processes affected in Gβ mutants are actually regulated by Gα (Ullah et al., 2003). In general, those phenotypes shared by Gα- and Gβ-deficient mutants are most likely due to disruption in processes mediated by Gα, while disruption of processes mediated by Gβ results in different or even opposite phenotypes (Ullah et al., 2003). Therefore, to avoid complications in interpretation, we chose processes with predominant Gβ signaling, namely, resistance against necrotrophic pathogens (Llorente, et al., 2005; Trusov, et al., 2006), auxin-regulated lateral root development (Ullah et al., 2003), and d-glucose inhibition of germination (Ullah et al., 2002; Chen et al., 2006b; Pandey et al., 2006; Wang et al., 2006).

Involvement of Gβγ1 in Resistance against Fungal Pathogens

Quantitative and in situ gene expression studies in transgenic Arabidopsis reporter lines using two different pathogens gave the first indication of the involvement of Gγ1 along with Gβ in the defense mechanisms against necrotrophic fungi. These observations were confirmed by the fact that the Gβ-deficient mutant agb1-2 and all of the mutants lacking AGG1 (agg1-1c, agg1RNAi, and agg1 agg2) showed increased susceptibility to F. oxysporum, with no statistically significant differences observed between them. The increased susceptibility of Gγ1-deficient mutants to F. oxysporum was shown for Col-0 and Ws. The slight increase in resistance observed for Gα-deficient mutants suggests that, in defense-related processes, Gα acts by sequestering the Gβγ1 dimer to the inactive heterotrimeric complex, thus effectively lowering the free available Gβγ1 pool (Llorente et al., 2005; Trusov et al., 2006). This is consistent with the finding that the expression of GPA1 is not altered by pathogen exposure (Y. Trusov and J.R. Botella, unpublished data). Even though A. brassicicola and F. oxysporum are both necrotrophic fungi, their infection mechanisms are different. As for F. oxysporum, the responses of all AGG1-deficient mutants and agb1-2 to A. brassicicola were statistically indistinguishable, being more severely affected than in the wild type. This finding suggests that the complete Gβγ1 dimer is required for defense. By contrast, mutants deficient in AGG2 but not AGG1 (agg2-1 and agg2RNAi) showed a wild-type phenotype in their behavior against both pathogens, thus precluding any significant role of the Gβγ2 dimer in pathogen resistance.

The susceptibility data are consistent with the molecular observations showing reduced induction of the plant defensin PDF1.2 by A. brassicicola in agb1-2 and all mutants lacking AGG1 (agg1-1c, agg1RNAi, and agg1 agg2) but wild-type induction in agg2 mutants. In addition, all AGG1-deficient mutants showed reduced responses to MeJA (statistically indistinguishable from Gβ-deficient mutants), supporting the hypothesis that MeJA signaling could be the link between G proteins and the defense response (Trusov et al., 2006).

Regulation of Lateral Root Development by Gβγ1- and Gβγ2-Mediated Signaling

In the young Arabidopsis primary root, auxin transport occurs acropetally through the stele tissue from the first true leaves, where it is primarily synthesized (Bhalerao et al., 2002). This auxin stream initiates early lateral root primordia (Reed et al., 1998; Bhalerao et al., 2002) and augments root-mediated auxin synthesis (Ljung et al., 2005). At a later stage, the root meristem synthesizes auxin, which moves up from the root tip through the epidermis (Mitchell and Davies, 1975; Tsurumi and Ohwaki, 1978; Jones, 1990, 1998; Rashotte et al., 2001), influencing lateral root initiation (Bhalerao et al., 2002; Ljung et al., 2005). Thus, auxin in both streams initiates lateral root formation, but different signaling mechanisms had not been distinguished previously.

We showed that AGB1, AGG1, and AGG2 are each expressed in roots, with AGB1 expression being observed in the stele, cortex, and epidermis, whereas AGG1 expression is restricted to the stele and AGG2 is predominantly active in the cortex and epidermis. Interestingly, none of the genes was expressed in lateral root primordia or in pericycle cells, which become the initials to lateral root meristems. Gβ attenuates auxin signaling during lateral root formation (Ullah et al., 2003), and we extended this finding by showing the Gγ subunits provide specificity in this response. While both AGG1 and AGG2 are involved in the inhibition of auxin-dependent lateral root initiation and both possible dimers, Gβγ1 and Gβγ2, exert a synergistic effect in auxin signaling attenuation, neither Gβγ dimer type is able to compensate for loss of the other. A likely explanation is that each dimer acts on different branches of the auxin/lateral root pathway. This duality does not occur in hypocotyls, as Gβγ1, and not Gβγ2, attenuates auxin-induced adventitious roots in the hypocotyl.

Considering that AGG1 is expressed in the root stele, where acropetal auxin transport occurs, while AGG2 is expressed in the cortex and epidermis, which are known to accommodate basipetal auxin transport, we hypothesized that Gβγ1 and Gβγ2 could be specifically involved in signaling for each of the two auxin streams. Consistent with this, we found that inhibition of acropetal auxin transport at the shoot–root junction affected agg1 mutants, while agg2 mutants were more responsive to the inhibition of basipetal auxin transport arising from the root tip. Furthermore, support for our hypothesis was provided by studying the gravitropic response, a process that is dependent on basipetal auxin transport. The reduced responsiveness of agb1-2 and the agg2 mutants is consistent with a signaling role for basipetally moving auxin in the root. Taking into account the localization of the proteins, we speculate that Gβγ1 could mediate internal signals while Gβγ2 could be involved in external/environmental signaling. Brassinosteroids and ethylene are logical candidates to be such internal signals, since both brassinosteroids and ethylene signal transduction pathways are influenced by heterotrimeric G proteins at various stages of plant development (Ullah et al., 2002) and there is evidence that brassinosteroids and ethylene promote lateral root development by increasing acropetal auxin transport (Bao et al., 2004) and by increasing auxin content locally at pericycle founder cells (Aloni et al., 2006). On the other hand, it is well known that a wide range of soil characteristics, such as availability of water or nutrients, can dramatically affect lateral root development (Vanneste et al., 2005). Signaling from one or more of these factors could be coupled by Gβγ2.

Germination and G Protein Signaling

The role of G proteins in seed germination is intriguing and complicated, since these proteins affect gibberellic acid, abscisic acid, brassinosteroids, MeJA, ethylene, and auxin signaling (Ashikari et al., 1999; Ueguchi-Tanaka et al., 2000; Wang et al., 2001; Ullah et al., 2002; Lapik and Kaufman, 2003; Chen et al., 2004; Pandey et al., 2006) as well as d-glucose sensitivity (Ullah et al., 2002; Chen et al., 2006b; Pandey et al., 2006; Wang et al., 2006). The gpa1 and agb1 null mutants show a number of alterations in seed germination, suggesting that GPA1 and AGB1 are involved in this process, although their specific roles are not known (Ullah et al., 2002; Chen et al., 2006b; Pandey et al., 2006). Here, we focused on traits dependent on Gβ-mediated signaling to establish the specificity of the Gγ subunits. The d-glucose hypersensitive phenotype of the Gβ null mutants is more severe than that for the Gα null mutants, implying that the predominant signaling element in d-glucose–regulated germination is the Gβγ dimer (Pandey et al., 2006; Wang et al., 2006). Our results indicate that both Gβγ1 and Gβγ2 dimers mediate this response, although their involvements are different. Gβγ1 is mostly involved in the osmotic component of the glucose response, although involvement in glucose signaling cannot be discounted, while Gβγ2 plays a role in glucose signaling but not in osmotic stress. The apparent contradiction of our results with the previously reported wild-type sensitivity of agb1-2 to a different osmotic agent, sorbitol (Pandey et al., 2006), can be explained by the masking effect that light intensity (used in that study) has on osmotic response (cf. Figure 6E with 6D). These data further illustrate the complexity of the germination process, implicating at least two independent signaling pathways involving Gβγ1 and Gβγ2 dimers and the additional effect of light intensity.

The fact that AGB1- and AGG1-deficient mutants are hypersensitive to osmotica raises the attractive possibility of the involvement of Gβγ1 signaling in osmoregulation (Zhu, 2002). The high expression levels observed for AGB1 and AGG1 in hydathods, highly specialized osmoregulatory organs, also suggests such a speculation.

γ Subunits Provide Functional Selectivity to the Gβγ Dimer

There are substantial similarities, but also important differences, between animal and plant heterotrimeric G proteins. They are structurally similar, suggesting a conserved mechanism of action (i.e., once a G protein–coupled receptor is activated, the associated G protein will dissociate and transduce the signal to downstream effectors through two functionally distinct subunits, Gα and Gβγ). However, plant G proteins lack the multiplicity of genes encoding each of the subunits, as in animals. It is this multiplicity that provides numerous combinatorial possibilities to the whole heterotrimer in order to mediate the action of hundreds of receptors in animal systems. Having single Gα and Gβ subunits begs the question of how plant G proteins are involved in a large variety of plant processes (Jones, 2002; Assmann, 2004; Jones and Assmann, 2004). The existence of two different Gγ subunits provides functional diversity to the entire heterotrimer for effector activation and receptor specificity. The similarities of the phenotypes displayed by Gβ- and Gγ-deficient mutants provide a functional association between the Gβ subunit and each of the Gγ subunits in plants, showing that both Gγ subunits form functional Gβγ dimers. We also showed that the two Gγ subunits serve independent, redundant, or complementary roles in planta, depending on the process and the tissue being studied. In some processes, such as defense against necrotrophic fungi, only one Gγ subunit is involved (AGG1). In other processes, such as auxin signaling and the development of lateral roots, both subunits are involved but are mechanistically different in their operation. In other processes, such as germination, both Gγ subunits are involved but with independent roles, with AGG2 implicated in glucose signaling and AGG1 mediating the response to osmotica (Figure 8).

Figure 8.
Two Arabidopsis Gγ Subunits Provide Functional Selectivity to the Gβγ Dimer.

In summary, the differential behavior of the Gγ mutants in known Gβ-mediated response pathways demonstrates that Gγ subunits provide functional selectivity to the plant heterotrimeric G proteins, providing a mechanism underlying the complexity in G protein–mediated signaling in plants.

METHODS

Plant Materials

The agg1-1 mutant allele of AGG1 in the Ws ecotype of Arabidopsis thaliana was generated and provided by the Institut National de la Recherche Agronomique (Versailles) (FLAG flanking sequence tag number 197F06) (Brunaud et al., 2002; Samson et al., 2002). The AGG2 allele agg2-1 in the Col-0 ecotype was obtained from the Salk Arabidopsis T-DNA mutant collection (Alonso et al., 2003) (SALK_010956). For each line, homozygous plants were selected using a three-primer PCR approach. PCR products across the insertion points were sequenced to confirm the exact position of the T-DNA.

The agg1-1 allele was introgressed into the Col-0 background by crossing agg1-1w with wild-type Col-0 plants and the hybrids backcrossed to wild-type Col-0 for eight successive generations. Isolation of the hybrids and backcrosses carrying the agg1-1 allele was performed by selecting for BASTA resistance conferred by the BAR gene present on the T-DNA (Samson et al., 2002). The final mutant line was designated agg1-1c. The double agg1 agg2 mutant was obtained by crossing agg1-1c with agg2-1. Plants carrying both homozygous alleles were identified from the segregating F2 population using BASTA selection and PCR analysis.

AGG1 and AGG2 RNAi constructs were generated as follows. An ~400-bp cDNA fragment for each of the genes was amplified by PCR using elongase (Invitrogen) and the following primers: for AGG1, 5′-CTCGAGGAATTCCTCTCTCTGACGTTGTCAGATC-3′ and 5′-ATCGATTGGTACCCATGTAAAATGATATCCTAGC-3′; for AGG2, 5′-CTCGAGATCTAGAGATGGAAGCGGGTAGCTCAA-3′ and 5′-AAGCTTGGATCCCCAATTACATCAAATTCACTG-3′. Restriction sites (underlined) were added at the ends of each primer for cloning into the pKANNIBAL vector (Wesley et al., 2001). Subsequently, the hairpin cassette was cloned into the binary vector pUQC477 obtained from Bernard J. Carroll (University of Queensland, Australia). Arabidopsis plants (Col-0 ecotype) were transformed by floral dipping (Clough and Bent, 1998). Primary transformants were selected with BASTA. Fifteen and 12 independent transgenic lines were obtained for agg1RNAi and agg2RNAi, respectively, and analyzed by RNA gel blot hybridization for downregulation of the corresponding genes. Lines with no detectable levels of mRNA were subjected to RT-PCR to confirm the lack of detectable message.

The promoter regions of AGB1, AGG1, and AGG2 were amplified from wild-type Arabidopsis (Col-0 ecotype) genomic DNA using the following primers: for AGG1, 5′-CACCGCCGAGGAATCGATCTGGCAT-3′ and 5′-TTGCAGAAAAATGCCAAAACGCCCAA-3′; for AGG2, 5′-CACCCTTGGCTCGTACTTCGAT-3′ and 5′-CAAAATTTCTCGAATTCAACCCTCA-3′; for AGB1, 5′-AACTCGAGTTACAAGCGAGCTTG-3′ and 5′-TTGGATCCATTCCGGGATCAGACTTAGGCTTC-3′. Restriction sites (underlined) were added at the ends of each primer for cloning purposes. Primers were generally designed to amplify the 5′ upstream region of each gene starting immediately upstream of the start codon. AGG1:GUS and AGG2:GUS lines were generated as described by Chen et al. (2006c). The AGB1 promoter fragment was cloned into pGEM-T Easy vector (Promega) and then transferred using XhoI and BamHI into the pAOV-intron-GUS vector (Mylne and Botella, 1998). The constructs were transformed into Arabidopsis (Col-0 ecotype) by Agrobacterium tumefaciens–mediated transformation (Bechtold et al., 1993). GUS staining was performed as described by Petsch et al. (2005).

Pathogen Preparation and Inoculations

Fusarium oxysporum (f. sp conglutinans) (BRIP 5176; Department of Primary Industries, Queensland, Australia) and Alternaria brassicicola (isolate UQ4273) were grown and plants were inoculated as described previously (Trusov et al., 2006).

Plate Assays

All plates contained 0.5× or 1× MS basal salts (PhytoTechnology Laboratories), 0.8% agar, and 1% sucrose unless stated otherwise. Stock solutions of MeJA and abscisic acid were added to autoclaved medium cooled to ~55°C at the designated concentrations. Seeds were sterilized in a 50% ethanol:1.5% peroxide solution and washed with sterile water or by incubation in a chamber filled with chlorine gas. After sowing, all seeds were stratified for 72 h at 4°C in darkness. Germination was determined as an obvious protrusion of the radicle. For root assays, seedlings were grown on vertical plates for 14 or 21 d, and the number of lateral roots was counted using a microscope. For gravitropic response assays, sterilized seeds were germinated and seedlings were grown vertically for 5 d under continuous light on square plates and then moved into darkness for another 24 h. Then, the plates were rotated 90° and left in darkness for 24 h. Seedlings were photographed and angle was measured from the digital images using NIH ImageJ software.

Isolation of RNA and Transcription Analysis

Total RNA for RNA gel blot analysis and RT-PCR was extracted as described previously (Purnell and Botella, 2007). Probes for RNA gel blots were labeled using the Rediprime II 32P radiolabeling kit (Amersham). Membranes were hybridized overnight in Church buffer (Church and Gilbert, 1984) at 65°C, washed twice in 0.1% SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate) and 0.1% SDS solution, and exposed to PhosphorImager plates for analysis (Molecular Dynamics). For RT-PCR, reverse transcription and PCR amplification were performed as described by Cazzonelli et al. (2005). PCR amplifications were performed using 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 as follows: agg1f, 5′-TGCGAGAGGAAACTGTGGTTTACG-3′; agg1r, 5′-CATCTGCAGCCTTCTCCTCCATTT-3′; agg2f, 5′-TGTATCCAACCAGTAACAAATGG-3′; agg2r, 5′-CGGCAGTGAATTTGATGTAATTG-3′. The ACTIN2 gene was used as a control for the RT-PCR experiments.

Accession Numbers

The Arabidopsis Genome Initiative identifiers for the genes described in this article are as follows: GPA1 (At2g26300), AGB1 (At4g34460), AGG1 (At3g63420), AGG2 (At3g22942), PDF1.2 (At5g44420), and ACT2 (At3g18780).

Acknowledgments

Work in J.R.B.'s laboratory is supported by Australian Research Council Discovery Grants DP0344924 and DP0772145. Work in A.M.J.'s laboratory on the Arabidopsis G protein is supported by the National Institute of General Medical Sciences (Grant GM-65989-01), the Department of Energy (Grant DE-FG02-05ER15671), and the National Science Foundation (Grant MCB-0209711).

Notes

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.plantcell.org) is: José Ramón Botella (ua.ude.qu@alletob.j).

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www.plantcell.org/cgi/doi/10.1105/tpc.107.050096

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