The Plant Cell, Vol. 14, S355-S373,
May 2002, Copyright © 2002,
American Society of Plant Biologists
Heterotrimeric and Unconventional GTP Binding Proteins in Plant Cell Signaling
Sarah M. Assmann1
Biology Department, Pennsylvania State University, 208 Mueller Laboratory, University Park, Pennsylvania 16802
1 To whom correspondence should be addressed. E-mail sma3{at}psu.edu; fax 814-865-9131
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INTRODUCTION
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Signal-transducing GTPases in plants include small G proteins, heterotrimeric G proteins, and, potentially, several unique types of GTP binding proteins that are not members of either of the aforementioned classes. This review will focus on recent discoveries concerning the roles of heterotrimeric and "unconventional" G proteins in plant cell signaling. Small G proteins are reviewed elsewhere in this issue (Yang, 2002 ). Plant heterotrimeric G proteins have been the subject of several other recent reviews to which the reader is also referred (Ma, 1994; Assmann, 1996; Hooley, 1998; Bischoff et al., 1999; Fujisawa et al., 2001). Ma's review (1994) does a particularly good job of summarizing the early, mostly biochemical and immunological, progress toward identifying plant G proteins, which will not be covered here.
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HETEROTRIMERIC G PROTEINS: THE MAMMALIAN PARADIGM
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Much of the conceptual framework for signaling by heterotrimeric G proteins has been provided by extensive research in mammalian systems. To provide a context and a framework for comparison, the mammalian paradigm (Figure 1)
is first reviewed briefly. Heterotrimeric G proteins are GTPases composed of  ,  , and  subunits. These GTPases are classically associated with plasma membrane receptors containing seven transmembrane domains (heptahelical receptors, 7-TMS receptors, or G protein–coupled receptors [GPCRs]). Receptor activation activates the G protein by inducing the exchange of GTP for GDP at a binding site on G. G and/or G then goes on to interact with effector proteins. Endogenous GTPase activity of G eventually returns the subunit to its inactive, GDP-bound form, resulting in reassociation of the trimer. In exceptional cases, the dissociation of G and G may not be obligatory for activation of the G protein (Klein et al., 2000).
Mammalian G subunits (39 to 46 kD) contain  20% highly conserved amino acids (Morris and Malbon, 1999). Gs also exhibit conservation of several important structural domains: a Ras-like GTPase domain, an -helical domain that influences the spontaneous GDP release rate, and an Asp/Glu-rich loop, which is involved in conformational changes upon GTP binding (Bourne et al., 1991). The presence of the latter two domains is one of the characteristics that distinguish G proteins from small GTPases. In mammals, 20 G subunits have been identified. Four classes of mammalian Gs exist, designated Gi, Gs, Gq, and G12/13 (Ma, 1994; Wilkie and Yokoyama, 1994). Gi and Gs were the initial members, identified by biochemical means, and their subscripts refer to inhibitory or stimulatory effects on the enzyme adenylate cyclase. Some species of G undergo lipid modifications that promote membrane association (Figure 2A)
(Casey, 1994).
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Figure 2. Lipid Modifiers and Effectors of G Proteins.
(A) Three types of G protein lipid modifications are shown. N-Myristoylation occurs by the attachment of the saturated fatty acid myristate (blue) to a conserved acceptor Gly (red) next to the initiator Met via a stable amino bond. In palmitoylated proteins, the saturated 16-carbon fatty acid palmitate (blue) is attached to Cys residues (red) via a labile thioester bond. Protein prenylation involves the attachment of the 15- and 20-carbon isoprenes farnesyl and geranylgeranyl (blue), respectively, to conserved Cys residues (red) at the C-terminal ends of proteins via a nonreversible thioester bond. The acceptor Cys residues are part of a conserved CaaX box motif in which C indicates Cys, a represents an aliphatic amino acid, and X is usually Ser, Met, Cys, Ala, Gln, or Leu. Ct and Nt indicate C- and N-terminal amino acid positions, respectively, relative to the acceptor amino acid. Proteolysis removes the final three amino acids, and the new C-terminal Cys is then prenylated. The Arabidopsis prototypical GPA1 contains a conserved myristoylation motif and a putative sequence for palmitoylation in the N-terminal region. The two Arabidopsis G protein subunits (AGG1 and AGG2) contain a prenyl group binding site in the C-terminal region.
(B) Cleavage sites and hydrolysis products of phospholipases regulated by G proteins. PLA2 is an acylhydrolase that specifically removes the acyl chain from the sn-2 position of the glycerol backbone (black). PLD and PI-PLC are phosphodiesterases that generate similar products, except that the phosphate group (blue) either stays with the lipid moiety (black) or goes with the head group (red). DAG, sn-1-palmitoyl,2-linoleoyl diacylglycerol; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; LysoPtdCho, sn-1-lysophosphatidylcholine; PtdCho, sn-1-palmitoyl,2-linoleoyl phosphatidylcholine; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdOH, sn-1-palmitoyl,2-linoleoyl phosphatidate.
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Mammals also possess 5 distinct G and at least 12 G subunits (Seack et al., 1998; Cook et al., 2001). A hallmark of the 35-kD G proteins is the WD-40 motif, consisting of seven or eight tandem repeats and a conserved Trp-Asp (WD) motif. These repeats assemble into a seven-bladed -propeller structure (Lambright et al., 1996; Sondek et al., 1996). G proteins range in mass from 7 to 10 kD and are not highly conserved. However, all Gs possess the C-terminal CaaX (where "a" is an aliphatic amino acid) site for isoprenylation (Figure 2A), which confers membrane association. A coiled-coil structure formed between G and G results in a noncovalent but very tight interaction between these two subunits, such that they function as a nondissociable dimer.
A nonexhaustive list of some G subunit effectors in mammalian systems includes adenylate cyclase, cyclic GMP phosphodiesterase, phosphoinositide 3-kinase, the phosphoinositide (PI)-phospholipase C (PI-PLC; Figure 2B) (Singer et al., 1997), phospholipase D (PLD; Figure 2B) (Singer et al., 1997), Na+/H+ exchange transporter (Voyno-Yasenetskaya et al., 1994; Voyno-Yasenetskaya, 1998), the TUBBY transcription factor (Santagata et al., 2001), and K+, Ca2+, and, to a lesser extent, Cl- and Na+ channels (Brown and Birnbaumer, 1990; Morris and Malbon, 1999; Reddy et al., 2001). G targets include phospholipase A2 (PLA2; Figure 2B), calcium channels, and some isoforms of adenylate cyclase and PI-PLC (Jelsema and Axelrod, 1987; Logothetis et al., 1987; Kim et al., 1989; Katz et al., 1992; de Waard et al., 1997; Morris and Malbon, 1999).
A nonexhaustive list of some ligands that interact with GPCRs to activate mammalian heterotrimeric G protein signaling includes light (Gt, members of the Gi class), odorants (Golf, members of the Gs subfamily), sweet and bitter molecules (Ggust, members of the Gi subfamily), and numerous hormones and neurotransmitters (many different Gs). Given this list, it is not surprising that mutations in G protein signaling components are responsible for numerous human genetic diseases. In addition, some bacterial diseases target G protein pathways. Indeed, the toxins from Bordetella pertussis (causative agent of whooping cough) and Vibrio cholerae (causative agent of cholera) are important experimental tools.
Cholera toxin (CTX)–induced ADP-ribosylation of an Arg residue on G inhibits its GTPase activity, resulting in persistent G activation. Although this residue is conserved among all Gs, only some types of Gs undergo ADP-ribosylation by CTX, possibly because of differential susceptibility to ADP-ribosylation factor (ARF). Pertussis toxin (PTX)–induced G ADP-ribosylation on a Cys near the G C terminus induces inactivation of signaling by interfering with G/receptor coupling. Other important experimental compounds include nonhydrolyzable GTPS, which locks G proteins in their active state, nonhydrolyzable GDPS, which promotes the inactive state, and the wasp venom mastoparan, which activates G proteins by mimicking the conformation of an activated receptor (Higashijima et al., 1988).
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PLANT HETEROTRIMERIC G PROTEIN SUBUNITS
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In contrast to mammals and invertebrates such as Caenorhabditis elegans, the Arabidopsis genome contains only one canonical G gene, GPA1 (Ma et al., 1990). GPA1 is 36% identical to Gis and transducins (Gts). It lacks the conserved ribosylation site for PTX but contains the sites for CTX and for N-myristoylation (Ma et al., 1990). Our sequence analysis (S. Coursol and S.M. Assmann, unpublished data) also suggests a possible palmitoylation site (Figure 2A). Homologs of GPA1 have been cloned from several dicots and monocots (Table 1) (Ma et al., 1991; Ma, 1994; Ishikawa et al., 1995; Gotor et al., 1996; Kusnetsov and Oelmueller, 1996b; Jones et al., 1998; Perroud et al., 2000). These proteins generally have 70 to 90% identity to each other and lesser identity (34 to 42%) to nonplant G subunits (Plakidou-Dymock et al., 1998).
GPA1 is a single-copy gene in Arabidopsis, but some polyploid species such as soybean have been shown to have two closely related GPA1 genes (Kim et al., 1995; Gotor et al., 1996). There is also a partial cDNA clone from wild oat aleurone, AfG1, that is only 40% identical to GPA1 and could represent a second class of plant G genes (Jones et al., 1998). Detailed enzymological characterization of Arabidopsis GPA1 has not been performed, although recombinant GPA1 has been shown to bind GTPS with a nanomolar dissociation constant (Wise et al., 1997). The rice G protein subunit, RGA1, has been confirmed to function as a G by classic biochemical tests, including those for binding specificity for GTPS over other nucleotides, specific GTPase activity, and ADP-ribosylation by CTX (Seo et al., 1995, 1997; Iwasaki et al., 1997).
GPA1 transcript and protein are found in all tissues except mature seed (Weiss et al., 1993), and GPA1 expression is particularly abundant in vascular tissue and actively dividing cells such as meristems (shoot, floral, and root), young embryos, and organ primordia (Weiss et al., 1993; Huang et al., 1994). A similar pattern has been observed for spinach SOGA1 transcript (Perroud et al., 2000). Rice RGA1 is highly expressed in internodes and florets (Fujisawa et al., 1999). Subcellularly, GPA1 has been immunolocalized to the plasma membrane and endoplasmic reticulum in both Arabidopsis and Nicotiana plumbaginifolia (Weiss et al., 1997; Kaydamov et al., 2000), whereas RGA1 has been immunolocalized in total membrane proteins (Seo et al., 1997). A putative G subunit also has been immunolocalized to the protonemal cell junctions of the moss Physcomitrella patens (Hutton et al., 1998), where it may regulate intercellular transport (Hutton et al., 1998) or Ca2+ channels (Schumaker and Gizinski, 1996).
The regulation of G gene expression by environmental factors has been examined in several species, although not yet in Arabidopsis. In N. plumbaginifolia, the synthetic auxin naphthylacetic acid increases NPGPA1 transcript expression in leaf discs, whereas gibberellins have no effect, abscisic acid (ABA) weakly reduces expression, and salicylic acid strongly reduces transcript levels (Kaydamov et al., 2000). Wounding and osmotic shock also have no effect on transcript levels. RGA1 expression in vegetative tissues is stimulated by light (Seo et al., 1995). Although transcriptional regulation of RGA1 by ABA has not been reported to date, the RGA1 promoter does contain an ABA-responsive element (ABRE) sequence (Seo et al., 1995). In hairy roots of tobacco, which have abundant root hairs resulting from infection by Agrobacterium rhizogenes, NtGPA2 transcript also is abundant (Ando et al., 2000), consistent with Arabidopsis GPA1 abundance in proliferating tissues. In spinach, circadian regulation of SOGA1 gene expression has been suggested (Perroud et al., 2000).
The Arabidopsis genome also contains one canonical G gene, AGB1, with 42% similarity to mammalian G subunits (Ma, 1994; Weiss et al., 1994), and homologs have been identified in maize, rice, wild oat, tobacco, and N. plumbaginifolia (Weiss et al., 1994; Ishikawa et al., 1996; Jones et al., 1998; Kusnetsov and Oelmueller, 1996a; Kaydamov et al., 2000). Arabidopsis and maize G transcripts are expressed in roots, shoots, and floral structures (Weiss et al., 1994). Expression of rice RG1 is higher in roots than in leaves (Ishikawa et al., 1996). NPGPB1 transcript levels closely follow the hormonal regulation described for NPGPA1, with the exception that ABA increases transcript abundance. NPGPB1 has been immunolocalized to the plasma membrane as well as the endoplasmic reticulum, in parallel with the localization of G (Kaydamov et al., 2000).
In tobacco leaves, G has been detected in both plasma membrane and isolated nuclei by protein immunoblot analysis (Peskan and Oelmueller, 2000), with abundance inversely correlated with leaf age. Tobacco G preferentially localizes to lipid rafts within the plasma membrane (Peskan et al., 2000). Lipid rafts are fleeting microdomains of unique lipid composition that are speculated to preferentially sequester certain proteins, thereby perhaps promoting "signalsome" association. Interestingly, mammalian Gi and Gs also were found recently to associate with lipid rafts, whereas Gq is concentrated in caveolae, which are cell surface invaginations that are rich in the protein caveolin (Oh and Schnitzer, 2001).
G genes cannot be identified on the basis of sequence homology alone. Recently, however, two Arabidopsis G subunit genes, AGG1 and AGG2, were identified by virtue of their interaction with a tobacco G protein in the yeast two-hybrid assay (Mason and Botella, 2000, 2001). The predicted G subunits are 48% identical to each other and display 24 to 31% identity with selected mammalian G subunits. AGG1 and AGG2 both possess certain common G characteristics, including small size (10.8 and 11.1 kD), a C-terminal CaaX motif, and an N-terminal region with the appropriate structure to interact with a G subunit. AGG1 and AGG2 also interact with Arabidopsis AGB1 in both yeast two-hybrid and in vitro binding assays (Mason and Botella, 2000, 2001). AGG1 and AGG2 transcripts are expressed in all tissues, with an expression pattern for the most part parallel to that of AGB1 (Mason and Botella, 2000, 2001).
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AUXINS, CELL PROLIFERATION, AND DEVELOPMENT
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Several early studies implicated G proteins in auxin signal transduction. Auxin was found to promote both GTPS association with rice coleoptile membrane vesicles and GTP hydrolysis by those vesicles, as would be predicted if auxin activated a G protein cycle (Zaina et al., 1990, 1991). Although not direct confirmation of a role for GPA1 in auxin response, recent studies are consistent with such a role. These studies identified two T-DNA mutant alleles of GPA1 and characterized their phenotypes (Ullah et al., 2001). The gpa1 null mutants exhibit normal root growth but show reduced cell division in both hypocotyls and leaves. Results obtained from the use of the mitotic reporter cyc1At-CDB-GUS in the gpa1 background are suggestive of prolongation of the G1 phase of the cell division cycle.
The high level of GPA1 expression reported in meristematic tissue is consistent with a role for GPA1 in cell division. Also consistent with this possibility is the observation that tobacco BY2 cells that overexpress GPA1 progress more rapidly through the cell cycle (Ullah et al., 2001). Interestingly, the expression of pea G subunits in yeast also promotes cell division (budding) of that organism (Marsh and Kaufman, 1999), and this occurs through a pathway independent of yeast mating pheromone signaling (which controls cell division via an endogenous G protein–dependent pathway). Thus, the role of G in regulating cell division may result from an interaction with downstream proteins that share a conserved function. One possible downstream target is PLA2, a known G protein effector in mammalian systems that mediates cell proliferation in many (but not all) cell types (Capper and Marshall, 2001). PLA2 is activated by auxin in soybean cell cultures (Scherer and Andre, 1989; Scherer, 1994), and auxin initiates more rapid progression through the cell cycle in cultured plant cells (Hobbie and Estelle, 1994).
gpa1 null mutants also exhibit an alteration in leaf shape to a more rounded, rotundifolia-type leaf. The rotundifolia3 mutant is deficient in a cytochrome P450 that may be involved in steroid biosynthesis (Kim et al., 1998). Ullah et al. (2001) note a reduced sensitivity to brassinolide in the gpa1 mutants, leading Ma (2001) to speculate that GPA1 also may play a role in brassinosteroid synthesis and signaling. Similarly, the G null mutant agb1-1 exhibits a rotundifolia leaf shape (Lease et al., 2001), suggesting that a functional G protein cycle may be important in the control of leaf shape. In addition, the agb1-1 mutant shows alterations in silique morphology similar to those conferred by the erecta mutation, including shorter, wider siliques with blunt tips and shorter, more tightly clustered floral buds.
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GIBBERELLINS AND ABA
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In keeping with the hypothesis that G protein–based signaling participates in the positive regulation of plant growth, heterotrimeric G proteins have been implicated in gibberellic acid (GA) responses in monocots. Recent breakthroughs in our understanding of the role of RGA1 in rice development have arisen from mutational analyses. The dwarfing mutation d1 maps to a G protein subunit gene, RGA1, as confirmed by allelism tests among several independent d1 mutants and by complementation. Antisense RGA1 rice plants phenocopy the d1 phenotype (Ashikari et al., 1999; Fujisawa et al., 1999), which includes not only reduced plant height as a result of reduced internode length but also broader, dark green leaf blades and sheaths, reduced elongation of panicles, and round grains (Ashikari et al., 1999).
In the germinating seed of grasses, GA plays an important role in stimulating secretion by the aleurone layer of hydrolytic enzymes such as -amylase that break down the endosperm, releasing resources that promote the growth of the young seedling. The d1 mutant was classified originally as GA insensitive based on the observation that GA application (10-8 M) did not result in -amylase production (Mitsunaga et al., 1994). In d1 mutant plants, 10-7 M GA only weakly stimulates expression of the -amylase gene itself and of the Myb transcription factor believed to activate this gene (Ueguchi-Tanaka et al., 2000; Fujisawa et al., 2001).
Consistent with this observation, earlier studies using wild oat aleurone had shown that Mas7 and GTPS promote expression of an -amylase–-glucuronidase (GUS) reporter gene and -amylase secretion, whereas GDPS opposes -amylase–GUS induction by GA (Jones et al., 1998). These researchers also demonstrated that G and the wild oat G gene AfG1 are expressed in the wild oat aleurone layer. A second gene expressed in this tissue, AfG2, also might encode a G subunit, although its sequence homology with known Gs is not as high (Jones et al., 1998). In addition to regulating the expression of -amylase, small or heterotrimeric G proteins (Wang et al., 1993) also may regulate its secretion, because GDPS inhibits Ca2+-stimulated exocytosis in barley aleurone protoplasts (Homann and Tester, 1997).
Despite the data favoring G protein involvement in GA signaling in aleurone, the G subunit does not appear obligatory for this response: in d1 rice, high (10-4 M) GA concentrations can stimulate the production of wild-type levels of -amylase activity, suggesting RGA1-independent as well as RGA1-dependent pathways of GA action (Ueguchi-Tanaka et al., 2000). In addition, and at first glance surprisingly, G protein activation also appears to play a role in inhibiting the aleurone GA response by ABA. One of the important downstream effectors of the ABA response is PLD, which produces phosphatidic acid and a head group from structural phospholipids (Figure 2B) (Pappan and Wang, 1999). In vitro, GTPS stimulates PLD activity in barley aleurone membranes, and GDPS and PTX each inhibits the ABA stimulation of this activity. PTX also reduces the ABA inhibition of GA-stimulated -amylase production (Ritchie and Gilroy, 2000).
Although identification at the molecular level of a heterotrimeric G protein involved in this response is still awaited, such a demonstration would be consistent with other studies that have provided pharmacological evidence for G protein regulation of PLD in the deflagellation response of Chlamydomonas eugametos and in PLD activation in carnation petals (Munnik et al., 1995). Surprisingly, however, recombinant tobacco GPA1 inhibits recombinant -type PLD in biochemical assays of PLD activity (Lein and Saalbach, 2001). This inhibition is reduced when NtGPA1 is bound with GTPS, and the authors speculate that perhaps G protein activation promotes the release of active PLD from the membrane.
PLD also is activated by ABA in stomatal guard cells (Jacob et al., 1999). Recent studies, although not yet showing a direct interaction of G and PLD in this system, provide convincing evidence that ABA signals through G proteins in this cell type as well. Experiments using the same gpa1 null lines used by Ullah et al. (2001) demonstrated that ABA inhibition of light-stimulated stomatal opening is eliminated in these genotypes (Wang et al., 2001). Likewise, ABA inhibition of the inward K+ channels that mediate K+ uptake during stomatal opening also is abolished.
However, not all guard cell ABA responses are eliminated: ABA stimulation of stomatal closure is normal. In patch-clamped guard cells from the gpa1 lines, ABA activation of anion channels that mediate malate2- and Cl- loss during stomatal closure occurs normally if cytosolic pH is allowed to change but is absent if cytosolic pH is strongly buffered. These results suggest that a pathway of ABA-stimulated anion channel regulation dependent on ABA-stimulated changes in cytosolic pH (Blatt, 2000) functions in parallel with, or as a backup to, a GPA1-dependent response during stomatal closure. This example of anion channel regulation by a G protein is unusual, given that only a few mammalian anion channels have been shown to be regulated by G proteins (Reddy et al., 2001).
The results with the gpa1 lines are consistent with an early study of G protein regulation of plant ion channels, in which it was shown that GTPS, mas7, and CTX inhibit inward K+ channels of guard cells, whereas GDPS promotes inward K+ channel activity (Fairley-Grenot and Assmann, 1991; Armstrong and Blatt, 1995). The effect of GTPS was prevented by buffering cytosolic Ca2+ to low concentrations, suggesting that ABA may act through GPA1 to increase cytosolic Ca2+. Biochemical studies (Lee et al., 1996) have shown that ABA activates PLC in guard cells, which produces inositol trisphosphate and diacylglycerol (Figure 2B); the former molecule releases Ca2+ from intracellular stores. Like ABA and GTPS, Ca2+ strongly inhibits the inward K+ channels (Schroeder and Hagiwara, 1989). In mammalian systems, G proteins function upstream of the -isoforms of PLC. It will be interesting to determine whether ABA activation of guard cell PLC is eliminated in the gpa1 lines, consistent with this model. In guard cells, ABA appears to signal, perhaps redundantly, through numerous secondary messengers, including not only PLC and PLD but also cyclic ADP-ribose and reactive oxygen species (McAinsh et al., 2000; Assmann and Wang, 2001; Schroeder et al., 2001). Regulation of these signals by GPA1 awaits investigation.
We also have observed the inhibition of inward K+ channel activity by GTPS, PTX, and CTX and the activation by GDPS in isolated patches of guard cell plasma membrane (Wu and Assmann, 1994) in experiments in which the solutions bathing both sides of the isolated membrane were provided by the experimenter. One would not expect changes in Ca2+ concentration to occur in this situation, unless Ca2+-permeable channels colocalize with the K+ channels and are activated by GTPS to provide a transient local increase of Ca2+ concentrations in the vicinity of the K+ channel. (In this regard, it is interesting that ABA activates guard cell Ca2+ channels in isolated membrane patches [Hamilton et al., 2000].) It is possible that GPA1 bypasses cytosolic secondary messengers to regulate the inward K+ channel either by direct physical interaction with the channel or through membrane-associated proteins or lipids (Brown, 1993). This theory is lent credence by the observation that ABA can activate outward K+ channels (Lemtiri-chlieh, 1998) in isolated membrane patches; a similar test needs to be performed for the inward K+ channel. The presence of both cytosol-dependent and membrane-delimited, functionally redundant pathways may indicate the importance of a "fail-safe" regulation of the guard cell response to ABA and drought.
There also is some pharmacological evidence consistent with a role for G protein activation in mediating stomatal opening (Lee et al., 1993; Kelly et al., 1995; Cousson and Vavasseur, 1998). Although these data have yet to be supported by genetic manipulations, a precedent for counteracting G proteins acting on the same process is well established. For example, in mammalian systems, Gs and Gi act in opposition to regulate adenylate cyclase. However, the paucity of plant G protein subunits suggests that this opposing regulation may derive in plants either from regulation via an unconventional G protein (see below) or from different "set points" of the cellular status of the guard cells that differentially dictate the outcome of G protein activation. For example, it has been demonstrated that guard cells rely on different osmotica to generate stomatal aperture changes dependent on the nature of incoming environmental stimuli (Poffenroth et al., 1992), suggesting that guard cell metabolism can exist in many different states. Likewise in the aleurone, there is evidence for G protein involvement in opposing pathways (see above).
Pharmacology also implicates G proteins in ion channel regulation in other types of plant cells. Thus, GTPS and CTX both reduce outward K+ currents in Vicia faba mesophyll cells, whereas GDPS enhances current magnitude and PTX has no effect (Li and Assmann, 1993). In mesophyll cells of this species, ABA reduces outward K+ current (Sutton et al., 2000), which is an effect analogous to that observed with GTPS and CTX. It will be interesting to determine whether this ABA response, like that of the inward K+ currents of guard cells, is affected in gpa1 knockout plants. In tobacco mesophyll cells, expression of the CTX A1 subunit reduces outward K+ currents, as does the overexpression of NtGPA1 sense RNA. However, overexpression of NtGPA1 antisense RNA has the identical result: a reduction in outward K+ current. These apparently conflicting results suggest the need to assess NtGPA1 protein levels in transgenic plants, an experiment that the authors report was technically problematic (Saalbach et al., 1999). Wegner and de Boer (1997) also have used pharmacological tools to implicate a G protein in a membrane-delimited pathway of activation of two inward K+ channels in xylem parenchyma, one of which shows instantaneous activation and the other of which shows slow, time-dependent current profiles.
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LIGHT RESPONSES
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Light signaling is one of the first pathways in which heterotrimeric G protein involvement was implicated in plants. A detailed biochemical study by Kaufman and colleagues demonstrated that a low fluence of blue light (<10-1 µmol m-2) excites GTPase activity in isolated plasma membrane–enriched fractions from apical buds of etiolated pea. The relevant G protein is thought to be a 40-kD membrane-associated protein that binds a GTP analog in a light-dependent manner, is recognized by transducin antibody, and is ADP-ribosylated by CTX and PTX (Warpeha et al., 1991). Subsequent research in pea has identified two G protein subunit cDNAs (Marsh and Kaufman, 1999); which of these is activated by blue light remains to be elucidated. Using similar assays, G protein mediation of blue-green light perception by rhodopsin in a flagellate green algae also has been proposed (Calenberg et al., 1998).
Other studies have implicated G proteins in cellular responses and gene regulation mediated by phytochrome. Early studies showed that the introduction of GDPS into the cytosol of wheat mesophyll protoplasts by electroporation inhibited protoplast swelling stimulated by red light. Conversely, the introduction of GTPS elicited swelling of protoplasts exposed to darkness or far-red light (Bossen et al., 1990). Red and blue light treatment of etiolated oat seedlings enhanced subsequent GTPS binding by plant extracts, and the red light effect was reversed by far-red light (Romero et al., 1991). CTX, which activates G proteins, upregulated CAB gene expression in etiolated oat seedlings and soybean cell cultures (Romero et al., 1991; Romero and Lam, 1993), mimicking the effects of red light on these transcripts.
Chua and colleagues (Neuhaus et al., 1993; Bowler et al., 1994a, 1994b) combined cell biological, molecular, and genetic approaches to more thoroughly address the role of heterotrimeric G proteins in phytochrome responses. They used the phytochrome A (PhyA)–deficient tomato mutant aurea to investigate whether G protein activation could initiate known phytochrome responses—chloroplast development, anthocyanin biosynthesis, and CAB gene expression—in a PhyA-deficient genetic background. Microinjection of purified oat PhyA into individual hypocotyl cells was shown to partially restore these responses in a cell autonomous and red/far-red light–reversible manner. Coinjection of either GDPS or PTX with PhyA eliminated the responses, whereas injection of GTPS alone initiated them. Analogous manipulations implicated calcium/calmodulin as acting downstream of the G protein. One caveat to this study is the low percentage (8% or less) of cells showing the responses described above. The extent to which this value may represent simply the difficulty of injecting a cell without damaging or killing it is not known.
Recently, Deng's group transformed Arabidopsis with constructs composed of a glucocorticoid-inducible promoter driving either GPA1 or this cDNA mutated to produce a constitutively active G (Okamoto et al., 2001). They concluded that G overexpression leads to hypersensitivity of hypocotyl growth inhibition by red, blue, and far-red light but does not alter responsiveness to exogenous GA application. PhyB is primarily responsible for red light–mediated inhibition, and in a PhyB genetic null background, the hypersensitivity to red light conferred by GPA1 overexpression is no longer observed. Similarly, PhyA primarily mediates far-red light inhibition of hypocotyl elongation, and GPA1 promotion of far-red light–induced hypocotyl inhibition was not observed when the GPA1 constructs were introduced into a PhyA mutant. However, the effect of GPA1 overexpression still occurred under blue light in a cry1 mutant, indicating that CRY1 is not a required photoreceptor for this blue light response.
These results indicating that active GPA1 inhibits hypocotyl elongation seem at odds with the results of Ullah et al. (2001), who reported that gpa1 null plants also exhibited a short hypocotyl phenotype. Furthermore, Okamoto et al. (2001) concluded from morphometric analysis that GPA1 overexpression affects cell elongation and not cell division, whereas Ullah et al. (2001) concluded that knocking out GPA1 affects hypocotyl length by reducing cell division without affecting cell elongation. One possibility, as Okamoto et al. (2001) acknowledge, is that the high levels of GPA1 overexpression confer upon this protein a function that it does not normally assume in wild-type cells. For example, high levels of overexpression of some mammalian Gs lead to an altered subcellular distribution of the subunit (Helms, 1995).
Another possible way to reconcile these observations is to assume that knocking out GPA1 or overexpressing GPA1 (particularly constitutively active GPA1) may result in an abundance of free G subunits, and perhaps it is these free G subunits that mediate the growth inhibition. However, this explanation does not reconcile the fact that one group reported an effect on cell division and the other group reported an effect on cell elongation. Dose-dependent auxin promotion of either cell expansion or cell division has been quantified in cultured plant cells and intact leaves (Chen et al., 2001a), and perhaps differential auxin sensitivity of the gpa1 null versus overexpressing lines is responsible for the divergent cellular phenotypes.
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PATHOGEN RESPONSES
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Fungal G proteins appear to be vital regulators of pathogenic status in plant fungal diseases (Choi et al., 1995; Liu and Dean, 1997; Regenfelder et al., 1997; Boelker, 1998; Yun et al., 1998; Coca et al., 2000). Conversely, evidence is accumulating for the importance of plant heterotrimeric G proteins in response to both bacterial and fungal pathogens. Thus, Beffa et al. (1995) showed that stable transformation of tobacco plants with the A1 subunit of CTX resulted in reduced susceptibility to Pseudomonas tabaci, accumulation of salicylic acid, and constitutive expression of pathogenesis-related genes.
A role of plant G proteins in plant responses to fungal pathogens was suggested by the work of Legendre et al. (1992), who showed that mastoparan elicits an oxidative burst in soybean cell suspensions, whereas CTX enhances the burst initiated by the elicitor Verticillium dahliae. They demonstrated subsequently that the elicitor polygalacturonic acid and the G protein activator mastoparan both stimulate PLC activity in this preparation, leading to an increase in inositol trisphosphate (Legendre et al., 1993).
Blumwald's group has demonstrated in tomato that race-specific elicitors from the fungus Cladosporium fulvum activate membrane redox reactions, leading to increased NADH oxidase activity and ferricyanide reduction in purified plasma membrane vesicles. Elicitor also stimulates plasma membrane H+-ATPase activity. GTPS or mastoparan application likewise initiates these responses, whereas GDPS prevents their elicitation (Vera-Estrella et al., 1994a, 1994b). H+-ATPase activation by elicitor occurs concurrently with a decrease in the transporter's phosphorylation status, and GTPS and CTX cause dephosphorylation as well. Elicitor treatment also dissociates from a multimeric complex a 42-kD protein that cross-reacts with G antibodies, providing biochemical evidence for elicitor activation of a G protein (Xing et al., 1997). In addition, Blumwald's group has published elegant data showing that a constitutively active version of tomato GPA1 increases the activity of a Ca2+ channel that they propose is involved in pathogen-mediated increases in cytosolic calcium (Aharon et al., 1998).
Elicitors also can induce the production of plant secondary metabolites. Roos et al. (1999) demonstrated that yeast elicitor triggers the formation of benzophenanthridine alkaloids in cultured cells of California poppy. Both elicitor and mastoparan activated a phospholipase activity, and PLA2 products such as lysophosphatidylcholine and linolenic acid enhanced alkaloid production in response to elicitor.
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TIP-GROWING CELLS
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Approaches using pharmacological regulators have been used to implicate heterotrimeric G proteins in root hair responses to rhizobial nodulation (Nod) factors, which initiate nodulation responses in legumes. ENOD12 is a gene whose expression is stimulated by Nod factor. Mastoparan stimulates expression of an ENOD12-GUS reporter; CTX has no effect but PTX opposes induction by Nod factor. Mastoparan also stimulates root hair deformation in Vicia sativa (den Hartog et al., 2001), possibly by reorganizing the actin cytoskeleton (Braun et al., 1999), and biochemical analyses suggest that both mastoparan and Nod factor stimulate PLD and PLC activity (den Hartog et al., 2001).
G proteins also have been implicated in the development of another tip-growing cell, the pollen tube (Ma et al., 1999). PTX inhibits tip growth in this system, whereas CTX stimulates pollen tube growth, as does microinjection of GTPS. A 41-kD plasma membrane protein from pollen tubes can be recognized by antibodies raised against conserved peptides of mammalian G subunits and can undergo ADP-ribosylation. Furthermore, pollen plasma membrane vesicles contain a GTPase activity that is stimulated by CTX. It will be helpful to conduct analogous experiments using Arabidopsis pollen tubes, given the well-characterized G protein complement and the wealth of mutants available in that species.
Ca2+ has been implicated as an important signaling component in both the Nod response and pollen tube growth. Nod factor elicits both Ca2+ spikes (Ehrhardt et al., 1996) and more stable increases in cytosolic calcium (Gehring et al., 1997), whereas pollen tubes exhibit well-characterized Ca2+ oscillations. Pharmacological tests have implicated PLC and Ca2+ as functioning downstream of mastoparan in the regulation of ENOD12 expression in the root epidermis (Pingret et al., 1998). Apparently, mastoparan does not elicit Ca2+ spikes in root hairs (Walker et al., 2000), but the possibility that G proteins regulate basal Ca2+ levels has not been evaluated. Similarly, no evaluation has been made concerning the effect of G protein regulators on Ca2+ oscillations in pollen tubes. However, Ma et al. (1999) present evidence that a heterotrimeric G protein could mediate pollen tube responses to extracellular calmodulin. Thus, the intriguing possibility is raised that G proteins might mediate extracellularly based as well as intracellularly based CaM signaling (Bossen et al., 1990; Neuhaus et al., 1993; Romero and Lam, 1993; Shiina et al., 1997).
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OTHER FUNCTIONS OF HETEROTRIMERIC G PROTEINS?
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The GPA1 subunit immunolocalizes to both the plasma membrane and the endoplasmic reticulum, whereas AGB1 is present in nuclear fractions. These unusual sites of G protein distribution suggest additional possible functions of plant heterotrimers. Heterotrimeric G protein subunits, and even GPCRs, have been localized to the nucleus in mammalian systems, in which functions in cell division, nuclear protein import, adipogenesis, and nuclear PLC and Ca2+ influxes have been posited (for review, see Willard and Crouch, 2000). At least one mammalian G subunit also has been localized to the rough endoplasmic reticulum, although it appears to play no role in protein translocation (Audigier et al., 1988). Other locales include the cytoskeleton, the trans-Golgi network, and the cytoplasm, where functions in vesicle trafficking and cytoskeletal rearrangement have been sought (Audigier et al., 1988; Helms, 1995; von Zastrow and Mostov, 2001; Zheng et al., 2001). Particularly given the dearth of prototypical GPCRs in the Arabidopsis genome (see below), the functions of GPA1 and AGB1 within the endoplasmic reticulum and nucleus are worth pursuing.
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UNCONVENTIONAL GTP BINDING PROTEINS
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Plants have several classes of "unconventional" GTP binding proteins whose signaling functions have only begun to be elucidated. One group of unconventional G proteins is the "extra-large G proteins," which in Arabidopsis comprise three genes: AtXLG1, AtXLG-like, and G-putative. AtXLG-like and G putative show 41 and 42% similarity to AtXLG1 at the amino acid level; G-putative, despite its name, is more similar to AtXLG1 than to GPA1. Only AtXLG1 has been characterized (Lee and Assmann, 1999). AtXLG1 is expressed throughout the plant and encodes a 99-kD protein with a C-terminal half harboring significant homology with G proteins, including motifs required for GTP binding as well as an Asp/Glu-rich loop and a predicted helical domain characteristic of Gs as opposed to small G proteins. Recombinant AtXLG1 protein binds GTP preferentially over other nucleotides, although the instability of the recombinant protein has thwarted efforts to assess its GTPase activity (Y.-R.J. Lee and S.M. Assmann, unpublished data).
It is interesting that AtXLG1 binds GTP, because it lacks some of the conserved amino acids thought from studies on mammalian systems to be critical for GTP binding. It is possible that the tertiary structure of the protein provides compensation for the functions of these residues. A unique aspect of AtXLG1 is its N-terminal half, which contains a Cys-rich region that is similar to zinc finger domains, and a TonB box. The TonB box is a consensus sequence found in proteins of the bacterial outer membrane involved in the transport of macromolecules such as vitamin B-12 and iron-chelator complexes.
Because of the presence of porins in the bacterial outer membrane, this membrane cannot build up an electrochemical gradient to drive active transport. The TonB box interacts with TonB proteins in the bacterial inner membrane, and this interaction is hypothesized to mediate energy transfer to the outer membrane for use in macromolecular transport (Postle, 1993; Larsen et al., 1999). Whether the TonB box of AtXLG1 plays an analogous role awaits further characterization. The TonB box is not found in the other members of this gene family, and to date, AtXLG1 homologs have not been found outside the plant kingdom. We are currently identifying T-DNA insertional mutations of the XLG genes to characterize phenotypes associated with XLG null mutants (L. Ding and S.M. Assmann, unpublished data).
Another large protein in Arabidopsis with potential GTP binding capability is RHD3 (root hair defective). Cloning of the RHD3 gene revealed that it encodes an 89-kD protein with two motifs that are conserved in GTP binding proteins (Wang et al., 1997). rhd3 mutants are so named because their initial characterization focused on the phenotype of short roots and short wavy root hairs with reduced vacuole size (Schiefelbein and Somerville, 1990). However, further study (Wang et al., 1997) showed that the RHD3 protein is important for cell expansion in shoots as well as roots, with hypocotyl cell length in the rhd3-1 mutant reaching only 45% of control wild-type lengths, a shoot phenotype similar to that seen in transgenic Arabidopsis lines with altered levels of GPA1 protein (Okamoto et al., 2001; Ullah et al., 2001).
Another set of interesting GTP binding proteins found in plants is the developmentally regulated G proteins (DRGs). Plant DRGs are expressed in all tissues at the mRNA and protein levels, with higher levels of transcript and protein found in actively growing tissues such as root apices and young stems (Devitt et al., 1999; Etheridge et al., 1999). The expression of pea and Arabidopsis DRG genes appears to be regulated by the cell cycle (Devitt et al., 1999), and Arabidopsis AtDRG1 protein has been immunolocalized to cytoplasmic vesicles, leading Etheridge et al. (1999) to speculate that DRGs may play a role in vesicle transport. The most striking feature of this class of G proteins is their remarkable sequence conservation, which spans archael and eukaryotic genomes (Mittenhuber, 2001). For example, AtDRG1 is >40% identical to archaebacterial members of this family. The strong evolutionary conservation of DRGs suggests a ubiquitous and fundamental function for this class of GTP binding proteins (Li and Trueb, 2000).
Two smaller G proteins also are worth mentioning: Arabidopsis ATGB1 (Biermann et al., 1996) and tomato ORFX at the fw2.2 locus (Frary et al., 2000). ATGB1 was identified in a screen for expression library clones that bound radiolabeled GTP. Despite its small size, phylogenetic analysis shows equal relatedness of ATGB1 to Gs and to the ARF/ARL/Sar superfamily. The function of ATGB1 remains unknown. fw2.2 was identified as a quantitative trait locus that regulates fruit size (Alpert and Tanksley, 1996). When the fw2.2 locus from a small-fruited tomato is introduced into a large-fruited variety, a decrease in fruit size occurs. ORFX, the gene responsible for the fw2.2 quantitative trait locus phenotype, encodes a product with no sequence homology with any protein of known function (Frary et al., 2000 |
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INTRODUCTION
HETEROTRIMERIC G PROTEINS: THE...