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American Society of Plant Biologists
A Mutant Arabidopsis Heterotrimeric G-Protein
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODSReferences |
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subunit. Therefore, elk4 was renamed agb1. agb1-1 plants express similar fruit phenotypes, as seen in er plants, but differ from er in that the stem is only slightly shorter than that in the wild type, the pedicel is slightly longer than that in the wild type, and the leaves are rounder than those in er mutants. Molecular analysis of agb1-1 indicates that it is likely a null allele. AGB1 mRNA is expressed in all tissues tested but is highest in the silique. Analysis of agb1-1 er double mutants suggests that AGB1 may function in an ER developmental pathway regulating silique width but that it functions in parallel pathways affecting silique length as well as leaf and stem development. The finding that AGB1 is involved in the control of organ shape suggests that heterotrimeric G-protein signaling is a developmental regulator in Arabidopsis. |
INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSReferences |
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; Venter et al., 2001). Furthermore, multiple genes encode each of three heterotrimeric G-protein subunits (
, , and 
) in animals, whereas Arabidopsis appears to have single genes encoding and subunits and two genes encoding subunits (Arabidopsis Genome Initiative, 2000; Mason and Botella, 2001; Venter et al., 2001). However, genes encoding G have low sequence similarity, and the presence of additional G genes in the genome cannot be excluded (Mason and Botella, 2000).
GPCRs interact with , , and heterotrimeric G-protein subunits (Hamm, 1998). During GPCR activation, the GPCR acts as a guanine nucleotide exchange factor, causing the subunit to exchange guanosine-diphosphate (GDP) for guanosine-triphosphate (GTP). Subsequently, -GTP separates from the dimer, and disassociation of all three subunits from the receptor occurs. Both -GTP and transduce the signal of the activated receptor to downstream effectors. The GTP on the subunit is hydrolyzed to GDP, inactivating the subunit and allowing its reassociation with to reform the inactive heterotrimer complex.
The definition of heterotrimeric G-protein subunits in plants is largely based on sequence similarities with animal heterotrimeric G-protein subunits. The Arabidopsis subunit GPA1 has 36% identity and 73% similarity with animal subunits (Ma et al., 1990), the subunit has 50% identity with some animal counterparts (Weiss et al., 1994), and Arabidopsis subunits show some sequence similarity with human subunits (Mason and Botella, 2000, 2001). In addition to sequence homologies, biochemical similarities between animal and plant heterotrimeric G proteins have been demonstrated. The rice subunit binds and hydrolyzes GTP (Seo et al., 1997). Furthermore, the and subunits are membrane localized (Weiss et al., 1997; Obrdlik et al., 2000), and the and subunits have been shown to bind to each other in vitro (Mason and Botella, 2000, 2001). However, it has not been demonstrated that the plant , , and subunits can form a trimer, nor has any subunit been shown to bind to any particular putative GPCR in plants.
Previously, the functions of heterotrimeric G-protein signaling in plants have been largely inferred from pharmacological studies (Wu and Assmann, 1994; Jones et al., 1998; Ritchie and Gilroy, 2000). These studies have suggested functions for heterotrimeric G-protein signaling in the regulation of ion channels, gibberellin signal transduction, abscisic acid signaling, as well as other possible functions.
Recently, loss-of-function mutants in the heterotrimeric G-protein subunits of rice and Arabidopsis have been described (Ashikari et al., 1999; Fujisawa et al., 1999; Ueguchi-Tanaka et al., 2000; Ullah et al., 2001; Wang et al., 2001). Although G-protein -subunit null mutants from both species are completely viable, they show several developmental defects. The rice mutant exhibits shortened internodes, rounded seeds, and partial insensitivity to gibberellin, whereas the Arabidopsis gpa1 mutants have rounded leaves and altered sensitivity to a number of phytohormones, including gibberellin. Furthermore, gpa1 affects either cell division or cell elongation, depending on the organ type. Moreover, abscisic acid–regulated inhibition of stomatal opening requires GPA1 function.
Together with the G null alleles, plant G overexpression studies have documented the importance of G-protein signaling (Okamoto et al., 2001; Ullah et al., 2001). Ectopic overexpression of GPA1 increased cell division, led to formation of adventitious meristems, and increased developmental sensitivity to low levels of light. In contrast to G, neither gain-of-function nor loss-of-function mutants for any plant heterotrimeric G-protein or subunits have been reported. Therefore, the relative physiological importance of these subunits has been unclear.
A genetic screen aimed at identifying genes functioning in the receptor-like kinase ERECTA (ER) signaling pathway was performed. The ER gene is predicted to encode a protein with 20 leucine-rich repeats in its extracellular domain, a single transmembrane domain, and an intracellular serine/threonine protein kinase domain (Torii et al., 1996). er mutants have pleiotropic phenotypes affecting the development of leaves, stems, flowers, and fruits (Rédei, 1962; Bowman, 1994; Torii et al., 1996). One of the mutants we identified in our screen encodes a mutant allele of the Arabidopsis heterotrimeric G-protein subunit (AGB1). agb1-1 exhibits several defects, including short, blunt fruits, rounded leaves, and shortened floral buds. The phenotypic characterization of agb1-1 demonstrates that heterotrimeric G proteins play a role in plant development and contribute to our understanding of plant cell signaling.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSReferences |
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; Bowman, 1994; Torii et al., 1996). In addition, the siliques of er mutants are shorter, wider, and have a blunt tip. To test our hypothesis, we screened ethyl methanesulfonic acid–mutagenized populations of Arabidopsis for the clustered flower phenotype, which we followed with observations of the silique morphology. As summarized in Table 1, the mutants were placed into five complementation groups and named elk1 through elk5 (for erecta-like). Each elk mutant was mapped, as summarized in Table 2. Because of a similar map position and phenotypes, complementation testing was performed between elk1 and transport inhibitor resistant3 (tir3) (Ruegger et al., 1997), which revealed that they are allelic. Therefore, elk1 alleles were renamed tir3-101 to tir3-110. The remainder of the elk mutants do not map near previously described mutants with similar phenotypes. As a first step toward ascertaining which mutants represent genes that may function with ER, we performed silique length measurements of elk mutants (Table 3). All of the elk loci, except elk4, have a shorter silique than does a complete loss-of-function er reference allele. Therefore, tir3, elk2, elk3, and elk5 mutants were not studied further because they likely function in either separate and/or additional developmental pathways, with ER controlling silique shape. However, elk4, represented by a single allele, has a silique length almost identical to that of er. Therefore, we performed more detailed studies with elk4. Upon molecular cloning of the ELK4 locus, elk4 was renamed agb1.
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; Torii et al., 1996). Stem length is slightly decreased in agb1 relative to the wild type, but it is not as short as in er, as shown in Figure 3B and Table 5. The agb1 er double mutant is shorter than either agb1 or er single mutants.
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50% as long as those in the wild type, whereas the lamina in agb1 is 125% wider and 85% as long as that in the wild type. These traits contribute to giving the agb1 rosette a more compact, rounded appearance than that of wild-type plants (data not shown).
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agb1 Encodes a Mutant Heterotrimeric G-Protein Subunit
Molecular identification of the agb1 mutant was performed by following a positional cloning strategy, as illustrated in Figure 5 . agb1 was found to map between the markers F3L17(4800) and nga1107 on the bottom arm of chromosome four. F2 plants that were recombinant for these flanking markers were assayed with additional markers internal to the first pair to define a smaller pool of recombinants. This process was repeated in an iterative manner until the genomic region encompassing the AGB1 locus was delimited to 61 kb. At this point there was one recombinant on the centromeric side of AGB1 and two recombinants at a marker telomeric to AGB1.
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subunit was able to complement agb1-1, as documented in Figure 6A
. The cDNA for this gene was previously identified and named AGB1, for Arabidopsis G 1 (Weiss et al., 1994). Thirty independent transformants were obtained with the AGB1-containing construct, all of which showed complementation. For example, the rounded leaf phenotype of agb1-1 was restored to its normal shape. Also, the blunt silique tip of agb1-1 was complemented, having the acute tip seen in wild-type fruits. DNA gel blot analysis confirmed the presence of the construct used to complement agb1 in the genome of complemented plants (data not shown).
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). Thus, agb1-1 is likely to be a null allele.
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DISCUSSION |
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; Bowman, 1994; Torii et al., 1996), which raises the possibility that AGB1 functions in a common silique developmental pathway with ER. This possibility was addressed by examination of agb1 er double mutants. The two simplest cases are that either AGB1 and ER function in parallel pathways or they function in a common developmental pathway. For example, if AGB1 and ER function in parallel pathways, the double mutant would be expected to show a more severe phenotype, evidenced by shorter, wider siliques. For this test to be meaningful, Arabidopsis fruits must have the capacity of being either shorter or wider than those seen in agb1 or er mutants. This requirement is satisfied because mutants have been reported that make the silique either shorter or wider than either agb1 or er. For example, tir3 mutants have siliques that are <60% as long as those in the wild type (Ruegger et al., 1997), and overexpression of CYP78A9 results in Arabidopsis fruits that are 40% wider than are er fruits (Ito and Meyerowitz, 2000). Statistical analysis of the agb1 er double mutant shows that the silique is significantly shorter than that in either the agb1 or er single mutant. Similarly, for leaf and stem phenotypes, the double mutants were more severe than were the er or agb1 single mutants. These observations suggest that AGB1 and ER function in parallel pathways that control the development of these traits. If they do function in parallel pathways, it is possible that agb1 simply phenocopies some aspects of er mutants but that each mutant has a different physiological basis for the similar aspects of their morphology.
However, statistical analysis of silique width, as well as tip angle measurements of agb1 er double mutants, showed these traits are not significantly different from those of either agb1 or er single mutant siliques. Futhermore, pedicel length in the double mutant is not significantly different from that in the er single mutant. These observations support the hypothesis that AGB1 functions in a common developmental pathway, with ER controlling these characteristics.
If AGB1 and ER function in a common developmental pathway, they might function in a common signal transduction cascade. Many signal transduction pathways modulate the expression of genes to bring about a cellular response (Wodarz and Nusse, 1998). One possibility we considered was that ER is transcriptionally activated by a GPCR pathway involving AGB1. If this case were true, we would expect to observe lower levels of ER expression in agb1-1. However, steady state levels of ER mRNA are similar in an agb1-1 background. A second possibility is that AGB1 expression requires functional ER. In this case, we would expect to see loss of AGB1 expression in an er background. This scenario is unlikely because steady state levels of AGB1 mRNA are only slightly reduced in er.
Another possibility is that AGB1 and ER may physically interact. To do so, two proteins must be co-localized. Both ER and AGB1 proteins are reported to be localized to the plasma membrane (Torii et al., 1996; Obrdlik et al., 2000). The possibility of interaction is further supported by the presence of a motif in ER that has been proposed to be important for interactions between the G subunit and its effectors, QXXER (Chen et al., 1995). In subdomain XI of the protein kinase domain of ER, a QXXDR sequence is present (Torii et al., 1996), suggesting the potential for interaction. However, in preliminary protein gel blot and glutathione S-transferase pull-down binding studies, we have not been able to detect a direct interaction between these two proteins. Other experimental approaches, which are closer to physiological conditions, may be required to detect an interaction. Alternatively, the genetic results we observed may not reflect a direct physical interaction between AGB1 and ER. Although the genetic results are tantalizing because they suggest a connection between receptor-like kinase and GPCR signaling pathways, the biochemical link, if one exists, remains to be determined.
Weiss et al. (1994) first cloned the AGB1 cDNA. They observed that AGB1 consists of seven WD-40 repeats and that it shows significant similarity to animal G subunits. However, numerous proteins contain the WD-40 repeat but are not heterotrimeric G-protein subunits (Arabidopsis Genome Initiative, 2000). For example, CONSTITUTIVE PHOTOMORPHOGENIC1 and TRANSPARENT TESTA GLABRA1 contain WD-40 repeats, but they are not G subunits (Deng et al., 1992; Walker et al., 1999). Does AGB1 encode a bona fide heterotrimeric G-protein subunit? Three lines of evidence from published literature support this hypothesis. First, by sequence homology, AGB1 shows 50% amino acid sequence identity to human G subunits 2 and 3 (Weiss et al., 1994). Second, a biochemical expectation of G subunits in animal and yeast cells is that the G subunit exists as a dimer with the subunit (Hamm, 1998). AGB1 can bind to Arabidopsis heterotrimeric G-protein subunits in vitro (Mason and Botella, 2000, 2001). Third, it was shown that AGB1 is membrane localized, which is consistent with the expectation of membrane localization (Hamm, 1998; Obrdlik et al., 2000). These observations indicate that AGB1 encodes a heterotrimeric G-protein subunit.
A question of central importance in interpreting the phenotypes of agb1-1 is whether it represents a loss-of-function allele, and if it does, whether it is a complete loss-of-function allele or only a partial loss-of-function one? When conceptually translated, agb1-1 is predicted to encode the N-terminal 35 amino acids, followed by 20 novel amino acids and a stop codon. This truncation eliminates 342 of the amino acids found in the wild-type protein and would result in loss of the WD-40 repeats that are required for G function (Hamm, 1998). Previous studies have also shown that a truncation mutant consisting of the first 41 amino acids of AGB1 cannot interact with the Arabidopsis G subunit (Mason and Botella, 2000). However, because an N-terminal portion of AGB1 is predicted to be expressed in agb1-1, it is possible that it represents a gain-of-function mutation. We think this is unlikely because gain-of-function mutations are typically dominant, whereas agb1-1 is completely recessive. Recessive mutants generally represent loss-of-function mutants. Because the predicted truncation of AGB1 occurs very close to the N terminus, and the mutant is recessive, it is likely that agb1-1 represents a null allele.
Little is known about GPCR signaling in plants. Mutant alleles of putative GPCRs in plants have not been reported. Although loss-of-function alleles of rice and Arabidopsis subunits have been described (Ashikari et al., 1999; Fujisawa et al., 1999; Ueguchi-Tanaka et al., 2000), no plant heterotrimeric G subunit mutants have been identified. agb1-1, a null allele of the Arabidopsis G 1 gene, is viable but causes an array of defects, including short, blunt fruits, rounded leaves, and shortened floral buds. This suggests that either GPCR signaling has a restricted role in normal growth and development or there are additional proteins that function as G that are not discernable by sequence homology.
In the mammalian GPCR signaling paradigm, the G complex associates with G-GDP (Hamm, 1998). This interaction terminates the ability of G to interact with other downstream effectors. If G is not functioning, as in the mutant backgrounds gpa1 or the rice G mutant dwarf1, the G dimer is predicted to be constitutively signaling. Therefore, G mutant phenotypes could be attributed to either an absence of G signaling or an overabundance of G signaling. On the other hand, if G is not functioning, as in the case of agb1, it is expected that the G cannot reassemble with a GPCR. In this scenario, the G will be unable to exchange GDP for GTP; as a result, all of the G is predicted to be "free" and in the GDP-bound inactive form. Thus, the phenotype of the G mutant agb1 may reflect a loss of both G and the G signaling pathways. The recent report (Ullah et al., 2001) of a null allele for the Arabidopsis G, gpa1, and the identification of agb1-1 offers the opportunity to genetically test these models.
Although a direct comparison of gpa1 and agb1-1 under the same growing conditions has not been made, we note some similarities between agb1 and the phenotypes documented for gpa1. Light-grown gpa1 has rounded leaves and shortened petioles (Ullah et al., 2001), similar to those observed in agb1. These leaf traits may reflect a G signaling pathway, because both G and G mutants appear to share this trait. On the other hand, the silique phenotypes of agb1 were not described in the initial description of gpa1. Therefore, these traits may reflect a G signaling pathway. A more conclusive determination of which physiological effects can be attributed to either G or G requires a direct side-by-side comparison and construction of the double mutant with gpa1 and agb1. Whatever the case may be, the identification and characterization of agb1 open up new avenues for investigation and contribute to a better understanding of plant cell signaling.
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METHODS |
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), five complementation groups were found. agb1-1 was backcrossed to wild-type Col two times before beginning genetic or morphological studies. agb1-1 segregates as a monogenic recessive in F2 crosses with Col (197 wild type: 55 agb1-1; 
2 = 1.35; df = 1; 0.3 > P > 0.2) (Russell, 1996). F1 progeny from crosses between agb1-1 and er-105 exhibit a wild-type phenotype. Wild-type Col and Wassilewskija (WS) ecotype seeds were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus). agb1-1 er-105 double mutants were constructed by crossing homozygous agb1-1 and er-105, allowing the F1 to self-fertilize, and generating a segregating F2. The double mutant was identified by choosing several plants with the er phenotype, backcrossing each plant to both er-105 and agb1-1, and finding an individual that complemented neither agb1-1 nor er-105.
Plant Growth and Measurements
Plants were grown under 100 µmol·m-2·sec-1 continuous white light at 23°C and fertilized weekly with Miracle Gro (Scotts, Port Washington, NY). The first five siliques from eight to 10 plants per genotype were used for measurement. The plants were 5 weeks old, and siliques were at stage 15/16 (Smyth et al., 1990), which are fully expanded siliques. The silique and pedicel lengths were measured by excising siliques and pedicels from the primary inflorescence, placing them on acetate sheets, photocopying, and measuring the photocopies using the software SigmaScan (Jandel Scientific, San Rafael, CA). Petiole length, lamina length, and lamina width were measured from leaf traces of the ninth leaf from at least 20 4-week-old plants per genotype by using SigmaScan. Lamina width was measured across the widest portion of the lamina. Inflorescence length of 5-week-old plants (n = 30) was measured with a ruler to the nearest millimeter. Silique width and the silique tip angle were measured with Metamorph (Universal Imaging Corp., Downingtown, PA) from digital images of siliques magnified with a dissecting microscope. Silique widths were measured at the lengthwise midpoint of each silique. The silique tip angle was determined by measuring the angle between a line tangential to the midpoint of the side of the silique and a line tangential to the apical tip of one of the two valves. Silique measurements represent the mean ±SD, with n = 40 to 50 organs measured for each genotype. For statistical analysis of quantitative data, Microsoft Excel was used to perform two-tailed Student's t tests assuming equal variances, with 0 for expected difference and = 0.05.
Microscopy
Scanning electron microscopy samples were fixed in 2.5% glutaraldehyde buffered with 0.1 M cacodylate, pH 7, postfixed with 1% osmium tetroxide, dehydrated through an ethanol series, critical point-dried, sputter-coated with platinum, mounted on stubs, and viewed with a Hitachi (Tokyo, Japan) S4700 FESEM.
Mapping
For mapping purposes, homozygous mutants were crossed with WS ecotype, the F1 was allowed to self-fertilize, and a segregating F2 population was generated. Recombinant inbred map positions for markers cited in Table 2 are based on the Lister and Dean (1993) map maintained on the Arabidopsis Information Resource website (Huala et al., 2001). Genomic DNA was isolated from 850 F2 plants from the mapping population that expressed the agb1 phenotype. SSLP and CAPS markers were used to map the elk loci (Konieczny and Ausubel, 1993; Bell and Ecker, 1994). Additional markers were generated during the course of mapping based on analysis of bacterial artificial chromosome (BAC) sequences for simple sequence repeats. The markers nga1107 and F3L17(4800), which flank the AGB1 locus, were tested with all 850 individuals. Those F2 individuals determined to be recombinant for the flanking markers were assayed with additional markers internal to the original flanking markers, to define a smaller set of recombinants. This process was repeated iteratively until the interval delimited by the markers encompassed 61 kb, spanning portions of the overlapping BAC clones F10M10 and T4L20. Nga1107 was analyzed on 4.5% agarose gels. All other SSLPs were analyzed by 32P-labeling the forward primer, separating the polymerase chain reaction (PCR) products on 5% denaturing polyacrylamide gels, and autoradiography. Marker names are a composite of the BAC sequence from which they were derived, followed by the approximate location in basepairs. The primers for the novel SSLP markers used in mapping are as follows: F3L17(4800): forward, 5'-ACATAACATGTTTGATCTAGCAC-3'; reverse, 5'-CTGCTTTTTGTT-CACACTGAACAT-3'; F4I10(34,400): forward, 5'-AAGGAAGAAGAAGACTGTTGAA-3'; reverse, 5'-CTCGTCCGTACCGTTCTCTTCC-3'; T16L1(40,200): forward, 5'-GGAAACTTAGATTGTAAAGCTTG-3'; reverse, 5'-AGCAGCAATCTCAGAGAAACATA-3'; F10M10(85,000): forward, 5'-CTGAATACAATATCTAATCTTTGA-3'; reverse, 5'-AAG-ACAAATATACAGTTTTCGACC-3'; T4L20(44,600): forward, 5'-CTAGACAAAAGAGAATTCAAAAGG-3'; reverse, 5'-TAAGGCAAAGTT-ACAAGATTACGT-3'; and T12J5(17,700): forward, 5'-ATCTCACTA-AATATTGACTAAGAG-3'; reverse, 5'-ATGTCTAGATTCCAATTG-TTTCA-3'.
Molecular Complementation of agb1
For brevity, only the construct that successfully complemented agb1-1 is described. The following primers, which include SalI and KpnI restriction sites at their 5' end to facilitate cloning, were used to generate a PCR product used for complementation: T4L20.4 forward, 5'-CTTGTCGACGGGAAAGCATGGATGAAGAAGATGAGCG-3'; and T4L20.4 reverse, 5'-CTTGGTACCGCTGTTCGTAAGGAGAATCAATGGGCT-3'. This construct includes 791 bp upstream of the initiation ATG and 1041 bp downstream of the stop codon. PCR was performed with the wild-type Col genomic DNA template; the PCR products were purified using Concert PCR purification kit (Invitrogen, Carlsbad, CA), digested sequentially with KpnI and SalI, and cloned into KpnI and SalI sites of pCAMBIA2300 (GenBank accession numbers AF234290–AF234316). This construct, pCAMBIA2300-T4L20.4, was transformed into Agrobacterium tumafaciens strain GV3101 via electroporation. Homozygous agb1-1 plants were transformed (Clough and Bent, 1998), the seeds were collected, and transformants were selected on medium containing half-strength Murashige and Skoog Salt Mix (Invitrogen, Carlsbad, CA), 50 µg·mL-1 kanamycin, and 1% agar.
Molecular Analysis of agb1-1
A genomic fragment was amplified from agb1-1 by using the same primers as those used for molecular complementation, and the coding portion of AGB1 was completely sequenced on both strands. The molecular lesion in agb1-1 is at nucleotide 15,169 of BAC T4L20 (GenBank accession number AL023094). Total RNA was isolated using the RNeasy midi kit (Qiagen, Valencia, CA). An AGB1 cDNA was isolated from agb1-1 by using reverse transcription–PCR with agb1-1 total RNA from leaves and flowers, using the following primers: forward, 5'-TCCGGTACCATGTCTGTCTCCGAGCTCAAAGAA-3'; and reverse, 5'-TCCGGTACCTCAAATCAC-TCTCCTGTGTCCTCC-3'. The cDNA was completely sequenced on both strands. For comparison of mRNA expression among genotypes, total RNA was isolated from rosette leaves of 3-week-old plants. To analyze AGB1 mRNA tissue-specific expression, total RNA was isolated from various tissue types of wild-type Col. For RNA gel blot analyses, 20 µg of total RNA was separated on a 1% MOPS-formaldehyde gel, the ethidium bromide–stained RNAs were photographed, and the RNA was transferred to a nylon membrane, cross-linked to the membrane by UV irradiation, hybridized with a 32P-labeled probe, and exposed to film for 2 days. AGB1 and ER probes were generated from a full-length cDNA template that was randomly primed. The relative level of expression was normalized by dividing the intensity of the ER or AGB1 signal by the intensity of the large rRNA from the ethidium bromide–stained gel. The result for each of the three genotypes was divided by the value obtained from the wild type to determine the relative difference in the level of expression in each mutant background compared with that of the wild type.
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Acknowledgments |
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Footnotes |
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Received July 30, 2001; accepted September 16, 2001.
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References |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSReferences |
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Subunit Affects Leaf, Flower, and Fruit Development
0.001). **Significantly different from the wild type (P 
0.057, Student's t test) or silique width (P 
