A role for G proteins in plant hormone signalling?

A role for G proteins in plant hormone signalling?

Richard Hooley

Institute of Arable Crops Research (IACR)-Long Ashton Research Station, Department of Agricultural Sciences, University ofBristol, Long Ashton, Bristol BS41 9AF, United Kingdom

(fax + 44 1275 394281; e-mail [email protected])

(Received January 20, 1999; accepted February 2, 1999)

Abstract — The G protein signalling pathway is one of the most highly conserved mechanisms that enables cells to sense andrespond to changes in their environment. Essential components of this are cell surface G protein-coupled receptors (GPCRs) thatperceive extracellular ligands, and heterotrimeric G proteins (G proteins) that transduce information from activated GPCRs todown-stream effectors such as enzymes or ion channels. It is now clear from a range of biochemical and molecular studies thatsome potential G protein signalling components exist in plants. The best examples of these are the seven transmembrane receptorhomologueGCR1and the Gα (GPA1) and Gâ (Gâ1) subunit homologues of heterotrimeric G proteins. G protein agonists andantagonists are known to influence a variety of signalling events in plants and have been used to implicate G proteins in a rangeof signalling pathways that include the plant hormones gibberellin and auxin. Furthermore, antisense suppression ofGCR1expression in Arabidopsis leads to a phenotype that supports a role for this receptor in cytokinin signalling. This review considersthe current evidence for and against functional G protein signalling pathways in higher plants and questions whether or not thesemight be involved in the action of certain plant hormones. © Elsevier, Paris

Auxin / cytokinin / gibberellin / heterotrimeric G protei n / G protein-coupled receptor

BA, benzyladenine / G protein, heterotrimeric G protein / GA, gibberellin / GPCR, G protein-coupled receptor / IAA,indole-3-acetic acid

1. INTRODUCTION

A long-standing objective of plant hormone biologyhas been to elucidate the molecular basis of signallingsystems that transduce hormone messages into thewide range of responses that they regulate. The lastfew years have witnessed substantial progress in ourunderstanding of plant intracellular signalling mecha-nisms and some of these have a direct bearing onhormone signal transduction. One emerging signallingpathway in plants is the G protein signalling cascade.This article reviews the evidence that higher plantshave some of the components of the well-characterisedG protein signalling pathway and considers to whatextent these components may or may not define a plantG protein signalling pathway that is involved inhormone signal transduction.

2. THE G PROTEIN SIGNALLING PATHWAY

The G protein signalling pathway is one of the mosthighly conserved membrane-located signalling mecha-

nisms and is responsible for transducing the action ofa wide variety of extracellular signalling mol-ecules [31, 43]. G protein-coupled receptors (GPCRs)are a primary component of this pathway and consti-tute a large and functionally diverse superfamily ofintegral membrane proteins. GPCRs specifically rec-ognise a huge range of ligands, these include olfac-trants, biogenic amines, visual pigments, neuropep-tides, a range of autocrine, paracrine and endocrinefactors, insect pheromones, fungal mating factors andchemotactic substances. The DNA and deduced aminoacid sequence of more than 700 GPCRs is known andthese all have seven stretches of between 20 and28 hydrophobic amino acids capable of forming atransmembraneα-helix. Thus, GPCRs are thought totraverse the membrane seven times forming a helicalcluster with associated extracellular and intracellularloops [43]. Receptors with these characteristics havebeen identified in vertebrates, invertebrates, arthro-pods, insects, nematodes, fungi, yeast, plants andviruses. Despite the diversity of species in which theyare found, and the range of agonists that stimulate

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Plant Physiol. Biochem., 0981-9428/99/5/© Elsevier, Paris

GPCRs, these receptors share considerable structuralsimilarity. This probably reflects conservation in theirmechanism of action that has been retained duringevolution.

When GPCRs bind their cognate ligand they un-dergo a conformational change that enables them tointeract with heterotrimeric GTP-binding proteins (Gproteins) [31, 42]. These G proteins consist ofα, â andγ subunits which, due to lipid modification of theαand â subunits, are associated with the cytoplasmicface of the plasma membrane. G protein activation byGPCRs stimulates the exchange of GDP for GTP in theguanine nucleotide binding site of the Gα subunit. Thiscauses conformational change in the Gα subunit thatpromotes dissociation from the Gâ/γ complex. Both theGTP-Gα subunit and the Gâ/γ complex are capable ofsignalling to effectors that include adenylyl cyclase,phospholipases, K+ and Ca2+ channels, and cyclic-GMP phosphodiesterase. The inherent GTPase activityof the Gα subunit restores the inactive heterotrimerwith bound GDP ready for another cycle of stimulationby the GPCR (figure 1).

3. PLANT HETEROTRIMERIC G PROTEINS

At the present time, there is no conclusive evidencethat a G protein signalling pathway operates in higherplants. However, it is becoming clear that higher plantsdo have some proteins that are similar to G proteinsignalling pathway components in other organisms.The first of these to be discovered were proteinssimilar to Gα subunits of G proteins and these wererapidly followed by Gâ subunits. The extent to whichthese might be functional counterparts of mammalianand other eukaryotic Gα and Gâ subunits, or for thatmatter components of a heterotrimer, is for the mo-ment unresolved [23]. Nevertheless, their occurrencein plants has spurred investigation of this question.

The first evidence that plants have Gα subunits camefrom biochemical and immunological studies (re-viewed by Ma [23]). Microsomal and plasma mem-branes from a range of species have been shown tocontain high affinity [α32P]-GTP- and [35S]-GTPγS-binding activity along with polypeptides that could beADP-ribosylated or which cross-reacted with antiseraagainst G protein Gα subunits, or Gα subunit peptides.However, as pointed out by Millner et al. [26], somereports should be treated with caution because theylack rigorous controls. Nevertheless, these observa-tions were later borne out by molecular studies thatidentified genomic and cDNA clones that encode for

proteins with many of the features characteristic of Gα

subunits (figure 2) from a number of plant species. Thefirst, GPA1, was identified in Arabidopsis [25] and is asingle copy gene [23] on chromosome 2, now knownto be about 10 kbp from Erecta (GenBank accessionAC004484). Homologues ofGPA1have been isolatedfrom tomato (TGA1) [24], Lotus japonicus(LjGPA1)[37], yellow lupin (LLGPROA) [20], spinach (SOG-PROALP) (GenBank accession Y16383), potato(STGPA1GEN) (GenBank accession X87836),Nicoti-ana tabacum (NTHTGPA) (GenBank accessionY08154) and Nicotiana plumbaginifolium (NPG-PROTSU) (GenBank accession Z72389). In otherspecies, two or more closely relatedGPA1-like se-quences have been identified. These include two se-quences in soybean (SGA1) [19], (SGA2) [9], foursequences in rice (RGA1) [13, 40] and two sequencesin pea (GenBank accessions U97043 and U97044).

A multiple alignment of the open reading framesencoded by these sequences illustrates that they areclosely related and probably represent a single class ofplant Gα subunits (figure 3). Gotor et al. [9] suggestedthat they should be placed in a new Gp family. Thesequences are 60 to 100 % identical at the amino acidlevel. Differences between them are minor, the mostobvious being a 30-amino acid deletion in one of theclasses of rice sequence, a 20-amino acid deletion inthe N. plumbaginifoliaclone, a potential alternativeinitiating methionine in theN. tabacumsequence anda different C-terminal motif in the rice sequences.These differences may be the basis of some functionaldiversity, certainly the differences in the C-terminalreceptor interaction region could be significant, al-though at present, there is no biochemical or physi-ological evidence to support this. The G1, G2, G3 andG4 regions of the aligned plant sequences show goodhomology to consensus motifs for Gα subunits [42].However, the G5 consensus motif of the plant se-quences differs from the majority of other Gα sub-units [42] perhaps reflecting differences in GDP/GTPbinding or GTPase activity between the plant andother eukaryotic Gα subunits. Pertussis toxin is a wellcharacterised G protein antagonist that ADP-ribosylates a C-terminal cysteine residue found inmany Gα subunits [31]. A number of investigatorshave found that pertussis toxin will interfere with plantsignalling [23] yet none of the plant Gα subunits havethe C-terminal pertussis-sensitive cysteine residue(figure 3). This suggests that other plant Gα subunitsmay exist but have not yet been cloned.

A second type of Gα subunit has recently beenidentified in Arabidopsis (GenBank accessions

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AF060941 and AF060942). This extra-large G proteinhas approximately 29 % amino acid identity to theplant GPA1 sequences but relative to these sequenceshas a 445-amino acid N terminal extension, a feature

found in the Xlαs G protein of rat [18]. The function ofthe Arabidopsis extra-large G protein is not knownalthough it suggests that plant G proteins may have arange of functions. Gâ subunit cDNAs have been

Figure 1. Diagram depicting stages in the G protein cycle.A, Ligand activated GPCR interacts with G protein stimulating GDP/GTP exchange;B, activated G protein dissociates into Gα subunit and Gâ/Gγ subunit complex. These can each interact with downstream effector molecules (E1and E2).C, Inherent GTPase activity of the Gα subunit hydrolyses the bound GTP and the heterotrimeric form is re-established ready to enteranother round of the cycle.

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cloned from maize, Arabidopsis and wild oat [15, 47].They have the seven WD-40 repeat regions character-istic of this class of signalling proteins and goodsequence similarity to a range of Gâ subunits fromeukaryotes. No Gγ subunit has been identified inplants, and, although there is a certain amount ofconfidence that one or more will turn up in the plantgenome databases [23], until this happens it cannot becertain that the Gα and Gâ subunits are part of aheterotrimer.

What is the function of these plant G proteinsubunits? The Gα subunits have between 36 and 42 %amino acid sequence identity to other eukaryotic Gα

subunits. This suggests a similar but by no meansidentical function. The differences in the G5 regionraised questions about the ability of the plant proteinsto bind and hydrolyse guanine nucleotides. However,these have recently been dispelled by the discoveriesthat recombinant Arabidopsis GPA1 binds GTPγS[49], recombinant rice GPA1 binds GTP, GTPγS andthe photoaffinity label 8-N3-GTP [14, 41] and thatrecombinant tomato TGA1 binds GTP [1]. In addition,GTPase activity of the recombinant rice and tomatoproteins [1, 14, 41] suggests that plant GPA1 proteinsare functionally related to Gα subunits. Nevertheless,the Kcat of GTP hydrolysis appears to be lower than

that of other eukaryotic Gα subunits [1]. The lowsequence similarity between plant and other Gα sub-units in receptor, effector andâ/γ interaction regionsmay simply reflect a requirement for correct secondarystructure conformation rather than primary amino acidsequence identity. Nevertheless, identifying proteinsthat interact with plant GPA1 is an important questionthat needs to be addressed experimentally.

Gα subunit expression has been studied in Arabi-dopsis and rice [41, 46]. Immunolocalisation of GPA1in Arabidopsis shows that it is present in roots, rosetteleaves, floral stems, cauline leaves, flowers and seedpods, though not in mature seed. The highest levels ofGPA1 occur in meristems, vascular tissue and imma-ture organs [46]. Immunolocalisation of the rice Gα

subunit RGA1 shows that it is more abundant in youngseedlings than mature plants. The highest levels werefound in seedling leaves and immature embryos whilethe lowest levels were in mature roots [41]. RGA1 isclearly present in rice plasma membrane [14], andboth Arabidopsis GPA1 and tomato TGA1 have beendetected predominantly in plasma membrane and en-doplasmic reticulum [1, 48]. This could indicate apotential role of these plant Gα subunits in signaltransduction events at the plasma membrane as well asin endomembrane trafficking.

Figure 2. Diagram depicting some of the primary sequence characteristics of G proteinα-subunits, based largely on Sprang [42]. The N and Ctermini of the polypeptide are indicated along with an approximate scale in amino acids. Conserved amino acid motifs in the five polypeptide loops(G1–G5) that form the guanine nucleotide binding domain are indicated as shaded blocks with the amino acid motif consensus underneath. Gα

subunits are lipid modified at their N terminus tethering them to the plasma membrane. This occurs through N-myristoylation at the G residue inthe characteristic N-terminal motif MGXXXS after removal of the initiating M, and by S-palmitoylation of C residues near the N terminus. Sitesthat are targets for ADP-ribosylation by cholera (CT) and pertussis (PT) toxins are indicated. These are present in some but not all Gα subunits.

396 R. Hooley


Figure 3. Multiple sequence alignment of plant Gα subunits. Shading indicates residues conserved between the sequences. Regions correspondingto the G1–G5 guanine nucleotide binding regions are indicated. The alignment was generated using the PileUp algorithm of the GCG package(version 8) with a GapWeight of 3.0 and GapLengthWeight of 0.1, and is displayed using GeneDoc 2.3.000 (http://www.cris.com/≈ketchup/genedoc.shtml) with a Blosum 62 score table. GenBank sequences for the following Gα subunits are compared: Arabidopsis (g166729),tomato (g170450), rice_1a (g2130075), rice_1b (g862310), rice_1c (g1346104), rice_1d (g600771), potato (g1771736),N. tabacum(g1552359),N. plumbaginifolium(g1749827), pea_1 (g2104771), pea_2 (g2104773), lupin (g1480298), lotus (g499078), soybean_1 (g439617), soybean_2(g1834453) and spinach (g3393003).


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4. PLANT G PROTEIN-COUPLEDRECEPTORS

Although a number of outstanding questions re-main, there is a good likelihood that higher plants havefunctional heterotrimeric G proteins that might beregulated by GPCRs. An attempt to couple humanGPCRs to plant G proteins was not successful al-though this might be a consequence of subtle differ-ences between the signalling mechanisms of theseparticular proteins [27]. Identification of the planthomologue of the GPCR superfamily GCR1 [16, 36]provided the first substantive evidence that this classof receptors predate the divergence of plants andanimals and identified a receptor that might potentiallyactivate GPA1. TheGCR1 cDNA encodes a 326-amino acid polypeptide that has up to 23 % amino acididentity (53 % similarity) to known GPCRs and anumber of amino acid motifs that are conserved insome classes of GPCRs. Hydropathy analysis indicates

that GCR1 has seven potential transmembrane span-ning domains and membrane topology prediction al-gorithms support a structure characteristic of GPCRs(figure 4).

5. PLANT G PROTEINS AND SIGNALTRANSDUCTION

Most efforts that have sought to define the functionof plant G proteins have relied on the use of pharma-cological agonists and antagonists. These include (a)the hydrolysis resistant GTP analogues, GTP-γ-S andGDP-â-S that respectively hold the Gα subunit inactive and inactive conformations, (b) the G proteinagonist Mas7 that mimics the third cytoplasmic loopof activated GPCRs [10, 11], and (c) the bacterialcholera and pertussis toxins that ADP-ribosylate spe-cific amino acids of certain classes of Gα subunit andeither activate (CTX) or inactivate (PTX) them. There

Figure 4. Diagram of the predicted membrane topology of GCR1 [36].

398 R. Hooley


is conclusive evidence that some of these agents affectthe activity of inward K+ channels implying that thistype of channel may be subject to G protein regulation.In guard cells, inward K+ channel activity is modu-lated by G protein agonists and antagonists in acomplex manner. Current interpretations of the dataare that multiple G protein signalling pathways prob-ably regulate inward K+ channels in guard cells [2].Guard cells are not the only cell types in which inwardK+ channels are G protein regulated. The activity oftwo classes of inward K+ channels in plasma mem-brane patches isolated from barley xylem parenchymaprotoplasts can be specifically stimulated by the hy-drolysis resistant G protein agonist guanosine 5’-[â,γ-imido]triphosphate [45]. In a more direct approachwith patch clamped plasma membrane, Aharon etal. [1] have demonstrated a stimulation in Ca2+ chan-nel activity by recombinant tomato TGA1 suggestingthat plasma membrane Ca2+ channels may also beregulated by G proteins in plants. Another moleculartarget of plant G proteins appears to be phospholipaseD [28, 29].

Similar types of investigations have suggested Gprotein involvement in blue and red light signalling [6,32, 38, 44], responses to fungal elicitors [21], patho-gen resistance and pathogen-related gene expres-sion [3], nodulation factor induction ofMtENOD12gene expression [35] and the transduction of planthormones [5, 15, 50].

5.1. G proteins and auxin signalling

The molecular basis of auxin signal transduction islargely unsolved although a range of evidence pointsto a plasma membrane-based signalling system forcertain auxin responses [30]. Some of the earliestevidence suggesting a possible involvement of Gproteins in plant hormone signalling came from stud-ies with auxin [50]. They observed that indole-3-aceticacid (IAA) increased the binding of [35S]-GTPγS torice coleoptile membrane vesicles while naphthyl-2-acetic acid, which has a lower biological activity thanIAA, did not effect [35S]-GTPγS binding. Using adifferent approach, Millner et al. [26] showed that asynthetic peptide that has auxin-like activity inViciafabaguard cells, and corresponds to the C-terminus ofmaize auxin-binding protein 1, stimulated [35S]-GTPγS binding to maize mesocotyl microsomes.These preliminary observations suggest that auxinaction might involve GDP/GTP exchange by a Gprotein; however, further data is required to consoli-date this. Zania et al. [50] also observed that GTPγSreduced [3H]-IAA binding to rice coleoptile membrane

vesicles and suggested that this might indicate that Gprotein activation inhibits IAA binding by a receptor.There has also been a report of a possible involvementof G proteins in swelling responses of protoplastsisolated from etiolated wheat leaves to auxin and otherhormones [5] though this has not been developedfurther.

An early event in auxin signal transduction appearsto be the activation of phospholipase A2 [33], althoughit is not yet clear how auxin activates this enzyme. Thefact that mastoparan and melittin can partially mimicauxin-stimulated elongation growth and activate phos-pholipase A2 suggests that a G protein may be in-volved; however, the possibility that these peptides areacting directly on the phospholipase A2 cannot bediscounted [39]. In summary, there is a possibility thatG proteins may be involved in auxin signalling,although at present the evidence is not entirely com-pelling.

5.2. G proteins and gibberellin signalling

Efforts to understand GA signal transduction haveconcentrated on the isolation and characterisation ofGA-response mutants and the GA induction of hydro-lase gene expression in aleurone cells [12]. In aleu-rone, evidence from two quite different experimentalapproaches suggests that GAs are perceived at theplasma membrane [12] and although identification of aGA receptor has not yet been accomplished, plasmamembrane located GA-binding proteins have beenidentified by GA photoaffinity labelling [22]. In GAtreated aleurone, events occur that may be componentsof GA signalling downstream of a plasma membranelocated GA receptor (reviewed by Bethke et al. [4]).These include an increase in cytoplasmic Ca2+ con-centration that appears to result from Ca2+ influx fromthe apoplast [7, 8]. This is followed by a transientincrease in cGMP [34] that appears to be necessary forGA-regulated gene expression. This leads to stimula-tion of the level of transcript for GAMyb, a transcrip-tion factor capable of transactivating gene expressionfrom the GA response element present inα-amylasepromoters.

Evidence supporting G protein involvement in GAsignalling has come from studying the effects ofvarious G protein agonists and antagonists in wild oataleurone protoplasts [15]. When wild oat aleuroneprotoplasts are incubated with Mas7 they produce andsecreteα-amylase in a dose dependent manner [15].Concentrations of Mas7 as low as 0.1µM gave asignificant response compared with untreated controls.Mas7 was found to induceα-amylase mRNA and


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drive the expression of anα-Amy2/54:GUSpromoter:reporter construct. It stimulatesα-amylase enzymeproduction and secretion with a virtually identical timecourse to GA and, like GA, the effect of Mas7 isovercome by ABA. The inactive mastoparan ana-logues, MasCP (control peptide), differing from Mas7by a single amino acid substitution, and Mas7-COOH,in which a free acid replaces the amine group at the Cterminus, do not induceα-amylase. Mas7 is thereforean effective GA mimic. Does it mimic GA by activat-ing a G protein or is it interacting with some othercomponent of GA signalling?

Jones et al. [15] addressed this question by studyingthe effects of the hydrolysis-resistant guanine nucle-otides GTP-γ-S and GDP-â-S on GA-induction ofα-Amy2/54:GUSexpression. These nucleotide ana-logues bind to Gα subunits and hold them in either theactive (GTP-γ-S-bound) or inactive (GDP-â-S-bound)form. GDP-â-S introduced into aleurone protoplastsduring transfection with reporter gene constructs com-pletely prevented GA1 induction of α-Amy2/54:GUSexpression, whereas GTP-γ-S slightly stimulated ex-pression. Taken together the effects of Mas7 andguanine nucleotide analogues on GA-induction ofα-amylase andα-Amy2/54:GUSexpression in wild oataleurone protoplasts suggest that a G protein or Gproteins are involved in GA signal transduction in thistissue.

Because GPA1 is not thought to be expressed inmature seeds [14, 46], Jones et al. [15] used PCR toclone a partial Gα subunit cDNA (AfGα1) and tworelated Gâ cDNAs (AfGâ1 andAfGâ2) from aleuroneof mature wild oat seed. Northern blot analysis con-firmed that these are expressed in aleurone cells. Thededuced amino acid sequence ofAfGα1 is 40 %identical to GPα1 [25]. The amino acid sequence ofone of the Gâ subunits,AfGâ1, is 91 % identical to themaize Gâ subunitZGâ1 [47].

6. GCR1 AND CYTOKININ SIGNALLING

Antisense suppression of the expression of the plantGPCR homologue GCR1 in transgenic Arabidopsisgave rise to a specific reduction in the sensitivity to thecytokinin benzyladenine (BA) [36]. Reduced sensitiv-ity was observed with a range of concentrations of BAin the inhibition of both root growth and hypocotyletiolation [36]. Responses to other plant hormoneswere unaffected by antisense suppression of GCR1.This suggests a role for GCR1 in either the perceptionor transduction of this plant hormone [36].

It is not known if GCR1 is a cytokinin receptor. Itmight instead be a downstream component in cytoki-nin signalling, or a receptor for a different ligand,whose signalling pathway interacts with cytokininsignalling. Another candidate cytokinin receptor isCKI1 that has sequence similarity to histidine-kinasetwo-component response-regulators [17]. Further re-search is needed to establish the function of, andrelationship between, GCR1 and CKI1 although anumber of possible models can be envisaged(figure 5).

Figure 5. Diagram depicting some of the possible relationshipsbetween GCR1 and CKI1.A, GCR1 and CKI1 may be receptors, ordownstream signalling components, in the transduction of differentcytokinin ligands; B, either GCR1 or CKI1 may be a cytokininreceptor in a pathway subject to crosstalk from a signalling pathwayinvolving the other protein;C, either GCR1 or CKI1 may be acytokinin receptor with the other protein as a downstream signallingcomponent.

400 R. Hooley


Although GCR1 has some of the characteristics of aGPCR, there is as yet no proof that it functions in amanner analogous to GPCRs. Therefore, it is impor-tant to determine not only the ligand that it binds butalso proteins with which it interacts. One candidate isof course the Arabidopsis Gα subunit homologueGPA1 but this needs to be tested experimentally.

7. SUMMARY

In summary, a range of biochemical and molecularevidence suggests that plants may have a functional Gprotein signalling pathway. Pharmacological and mo-lecular evidence argues that the action of three classesof plant hormones may be mediated, or stronglyaffected, by G protein signalling. Although persuasive,the present data is far from conclusive. Many furtherquestions need to be answered, not least of which whyG proteins are implicated in so many signalling eventsin plants, yet only a single GPCR homologue and atmost two Gα subunits have so far been identified inplants.

Acknowledgments

IACR receives grant-aided support from the Bio-technology and Biological Sciences Research Council(BBSRC) of the United Kingdom. This work wassupported by the BBSRC Intracellular Signalling pro-gramme, the BBSRC Integration in Cellular Re-sponses Programme and European Union FrameworkIV Programme.

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