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Study of the Constitutively Active Form of the α Subunit of Rice Heterotrimeric G Proteins

Study of the Constitutively Active Form of the α Subunit of Rice Heterotrimeric G Proteins Abstract We used site-directed mutagenesis to engineer two constitutively active forms of the α subunit of a rice heterotrimeric G protein. The recombinant proteins produced from these novel cDNAs had GTP-binding activity but no GTPase activity. A chimeric gene for a constitutively active form of the α subunit was introduced into the rice mutant d1, which is defective for the α-subunit gene. All the transformants essentially showed a wild-type phenotype compared with normal cultivars, although seed sizes were substantially increased and internode lengths also showed some increase. Heterotrimeric G proteins regulate the activity of downstream effectors following perception of external signals by receptors. Agonist-induced stimulation of G protein-coupled receptors causes heterotrimeric G proteins to dissociate into α subunits and βγ dimer subunits. In mammals, dissociated α subunits and βγ dimers transmit the receptor-generated signals to effectors in an antagonistic, synergistic or independent manner, depending upon the species of the three subunits (Clapham and Neer 1993). Progress in our understanding of the function of plant heterotrimeric G proteins has been advanced through analysis of mutants that have defects in the subunit genes (Ashikari et al. 1999, Fujisawa et al. 1999, Lease et al. 2001, Ullah et al. 2001). Mutants of the α-subunit gene isolated from rice and Arabidopsis were designated as d1 (Ashikari et al. 1999, Fujisawa et al. 1999) and gpa1 (Ullah et al. 2001), respectively. Studies on d1 have suggested the G proteins may be involved in more than three signaling pathways, including the gibberellin (GA) (Ueguchi-Tanaka et al. 2000), pathogen infection response (Suharsono et al. 2002) and light (Iwasaki et al. 2002) signaling pathways. d1 shows abnormal morphology such as dwarfism, dark green leaves and small round seed. The findings revealed that the G proteins are functional molecules regulating the body plans in rice. Studies on gpa1 raised the possibility that the G protein may be involved a number of signaling pathways, including those of GA, auxin, abscisic acid, ethylene, light and sugar (Ellis and Miles 2001, Ullah et al. 2001). Recently, it was proposed that sphingosine-1-phosphate is one of the signal molecules of the G protein-mediated signaling pathways in plants (Coursol et al. 2003). In addition, a putative G protein-coupled receptor (GCR1) (Pandey and Assmann 2004) and a regulator of G protein signaling (RGS) (Chen et al. 2003) have been shown to interact with the α subunits of heterotrimeric G proteins. Various proteins, for example cupin domain (Lapik and Kaufman 2003), calcium channel (Aharon et al. 1998) and a phospholipase D (Zhao and Wang 2004), have been proposed as effectors targeted by G proteins in plants. There is, however, little definitive information on the connection or relationship between putative signals, receptors and effectors in plants. In mammals, activators of G proteins, such as mastoparan, cholera toxin and GTPγS, and inhibitors of G proteins, such as GDPβS and pertussis toxin, are useful tools in the analysis of G protein-mediated signaling (Kaziro et al. 1991). However, mastoparan affects not only the G protein but also other proteins such as calmodulin and phospholipase C in mammals (Ross and Higashijima 1994). In addition, mastoparan 7, an active analog of mastoparan, was found to cause damage to cells in a G protein-independent manner in rice plants (Fujisawa et al. 2001b). Much controversy has arisen over whether the use of these activators and inhibitors is appropriate for the study of the G protein-mediated signaling in plants (Fujisawa et al. 2001a). An alternative approach that avoids such controversy is to convert the α subunits to constitutively active forms by amino acid substitution. Constitutively active α subunits regulate downstream effectors even in the absence of signals and stimulated receptors. As a result, constitutively active subunits are valuable tools for investigating G protein-mediated signaling in plants. In this report, we describe formation of constitutively active forms of the α subunit of rice heterotrimeric G proteins as a result of amino acid substitutions mediated by site-directed mutagenesis. We show that the constitutively active forms are able to rescue the abnormal phenotype of rice mutants defective in the α-subunit gene. It has been shown that exchange of only one amino acid in the sequence of α subunits of mammalian heterotrimeric G proteins is sufficient to result in a constitutively active form (Kaziro et al. 1991). On this basis, we introduced mutations into the α subunit (RGA1) of rice (Fig. 1). In one example, site-directed mutagenesis of RGA1 cDNA at position 223 caused the substitution of leucine (L) by glutamine (Q); the resultant clone was designated as Q223L cDNA. We introduced four mutations into RGA1 cDNA each with a different amino acid substitution at a different position, clones G48V, R191C, Q223L and A356S cDNAs (Fig. 1). Previously, we showed that a recombinant α subunit (RGA1) could be synthesized in a soluble form in Escherichia coli and purified (Iwasaki et al. 1997). In the present work, the site-directed mutagenized recombinant proteins (rG48V, rR191C, rQ223L and rA356S) were synthesized in E. coli and purified, according to the same procedures. All recombinant proteins were highly purified in water-soluble form, not as inclusion bodies in E. coli (Fig. 2A). When an α subunit is forced to become constitutively active through amino acid exchange, its GTPase and GTP-binding activities may be altered. Therefore, we first screened the GTPase activity of purified rRGA1, rG48V, rR191C, rQ223L and rA356S (Fig. 2B). Two of these, rQ223L and rR191C, had lost all GTPase activity, whereas the other two, rG48V and rA356S, still showed some GTPase activity albeit at much lower levels than in rRGA1. Next, we assayed the GTP-binding activity of the recombinant proteins (Fig. 2C). The proteins were incubated with [γ-32P]GTP and the amount of bound [γ-32P]GTP measured by liquid scintillation spectrometry. All four recombinant proteins were found to bind [γ-32P]GTP; however, they differed in their abilities to retain [γ-32P]GTP at longer incubation times. The [γ-32P]GTP-bound rQ223L and rR191C retained [γ-32P]GTP during further incubation. The [γ-32P]GTP-bound rG48V and rA356S lost γ-32P with time; they did not lose GTPase activity (Fig. 2C). Thus, rQ223L and rR191C keep the ability to bind GTP and lose GTPase activity. The results strongly indicate that rQ223L and rR191C are constitutively active forms of the α subunit. In vitro mutagenized α subunits of plant heterotrimeric G proteins have also been constructed for Arabidopsis (Okamoto et al. 2001) and tomato (Aharon et al. 1998). No biochemical data were reported for the Arabidopsis protein corresponding to Q223L of rice. The tomato protein corresponding to Q223L was fused to glutathione S-transferase; it appeared to have no GTPase activity but to have GTP-binding activity. However, no kinetic data were presented (principally because GTP binding was measured on the membrane after SDS–PAGE), and thus further analysis is necessary to confirm that the tomato protein truly lacks GTPase activity but has GTP-binding activity. Our use of purified proteins and detailed enzyme characterization has enabled us to conclude that both R191C and Q223L are constitutively active forms of the α subunit of the rice heterotrimeric G proteins. Part of the cDNA for Q223L was fused to the RGA1 gene to construct a chimeric gene, ProRGA1:QL (Fig. 3A). ProRGA1:QL is controlled by the promoter of the RGA1 gene and contains two introns in the RGA1 gene fragment. After the chimeric gene was fused to a binary vector, pBI-121Hm, it was introduced into calli of the rice mutant, d1, via the Agrobacterium transformation method. The dl mutant is defective in the gene of the α subunit of heterotrimeric G proteins and thus contains no α subunit. Hygromycin-resistant transformants were selected for Q223L expression by reverse transcription–polymerase chain reaction (RT–PCR). Mutant d1 plants show dwarfism, bear small round seeds, and form dark green leaves. In contrast, all rice transformants accumulating Q223L protein on the d1 background showed an essentially wild-type phenotype (Fig. 3B). The level of Q223L protein in the transformants was assayed using an anti-α subunit antibody. A typical Western blot, from the QL/d1 line of selected transformants, is shown in Fig. 3C. The constitutively active form of the α subunit accumulated in QL/d1 at the same level as seen in the normal cultivar. The unhulled seeds of transformants were about 5% longer than those of a normal cultivar, although unhulled seed widths were similar (Fig. 3D, Table 1). In transformants, unhulled seeds, hulled seeds and the hulls were approximately 20% heavier than those of normal cultivar rice (Table 2). Internode lengths of QL/d1 were about 5% longer than those of the normal cultivar (Table 3). Our data clearly show that constitutive activation of the α subunit by Q223L had a more substantial effect on seed formation than on internode length. Plant heterotrimeric G proteins are involved in multiple signaling pathways, including light, plant hormones and elicitor, as demonstrated by comparisons of signal responses of wild-type plants and G protein subunit mutants (Ellis and Miles 2001, Iwasaki et al. 2002, Jones and Assmann 2004). Transformants that have constitutive overexpression of the α subunit have been studied in Arabidopsis, oats and rice with the purpose of further elucidating the signal pathways through which heterotrimeric G proteins act. A study of transformants overexpressing the constitutively active α subunit (Q222L) in Arabidopsis suggested that the α subunit of heterotrimeric G proteins may act on a discrete branch of the phytochrome A signaling pathway; the α subunit seemed to be involved indirectly rather than directly with light signaling (Okamoto et al. 2001). In oats (Jones et al. 1998) and rice (Mitsunaga et al. 1994, Ueguchi-Tanaka et al. 2000), before discovery of G protein mutants, it was believed initially that the heterotrimeric G proteins were concerned with GA signaling. The identification of the rice dwarf mutant, d1, which has a mutation in the α subunit gene of the heterotrimeric G proteins, supported this perception. The mRNA level and enzymatic activity of α-amylase after application of GA3 were compared in embryo-less half-seeds of d1 and the normal cultivar. The d1 mutant gave a reduced response to low concentrations of GA3 compared with the normal cultivar, but a similar response at high concentration (Ueguchi-Tanaka et al. 2000). From these results, we considered that rice heterotrimeric G proteins were likely to be involved with GA signaling at low, but not high, concentrations of GAs (Iwasaki et al. 2002). There is no unequivocal evidence that G proteins directly regulate the GA signaling pathway through the transcriptional activation of GAMyb, a positive transcriptional factor of the α-amylase gene. It cannot be ruled out that the G proteins are indirect regulators of the GA signaling pathway. Lovegrove and Hooley (2000) proposed that such indirect regulation could arise through post-translational events after the perception of GAs. In the present work, rice dl transformants expressing Q223L did not show a slender phenotype but rather were essentially wild type (Fig. 3B). This suggests that it is unlikely that rice heterotrimeric G proteins are concerned with the major GA signaling pathways. Embryo-less half-seeds need GA for synthesis of α-amylase. If the constitutively active form of the α subunit was a positive regulator for major GA signaling, then it would be expected that α-amylase would be induced in embryo-less half-seeds of QL/d1 without GA. However, when we measured the activity of α-amylase in the embryo-less half-seeds of the transformants and normal cultivars (Mitsunaga et al. 1994), we found that the α-amylase content of QL/d1 was similar to that of the normal cultivar (data not shown). Consequently, we believe that heterotrimeric G proteins do not directly regulate the GA signaling pathway in rice. The present work shows that Q223L plays important roles in seed formation and thus we propose that Q223L would be a valuable tool to identify the G protein-mediated signaling pathways involved in seed formation. A rice normal cultivar (Oryza sativa L. cv. Nipponbare) and a rice dwarf mutant, d1 (DK22), were used in this study. The recurrent parent of d1 (DK22) was Nipponbare. All rice plants (normal cultivar, d1 and rice transformants) were grown under 14 h light, with cool white fluorescent light at 30,000 lux at 30°C, and 10 h dark at 25°C cycles. Site-directed mutagenesis was carried out using the Mutan-Express Km kit (Takara Bio Inc., Tokyo, Japan) in accordance with the manufacturer’s recommended protocol. The first step was to subclone the full-length sequence of RGA1 cDNA (GeneBank accession no. D38232) into the pKF19 plasmid. Four oligonucleotides were used in the site-directed mutagenesis experiments: G48V oligonucleotide (5′-TCCCTGATTCTACCGCACCAAG-3′); R191C oligonucleotide (5′-TTGTCCGTACACATGCATAAAGCAC-3′); Q223L oligonucleotide (5′-CTCATTCCTCAGGCCTCCTAC-3′); and A356S oligonucleotide (5′-CTGGTCTAGGCTCGTAGTTCTG-3′). All oligonucleotide sequences are shown in the antisense orientation of the RGA1 cDNA. The mutagenized constructs were sequenced to confirm mutagenesis and fidelity. The open reading frames of RGA1 and mutagenized relatives were subcloned, in-frame, in pQE30 vector (Qiagen K.K., Tokyo, Japan) and the pQE30 derivatives were transformed into E. coli BL21/DE3 with pREP4 plasmid. Recombinant proteins were synthesized in E. coli BL21/DE3 as described previously (Iwasaki et al. 1997). GTPase activity was assayed by the method, with minor modifications, described previously (Higashijima et al. 1987). [γ-32P]GTP (NEG-004Z, specific activity 222 TBq mmol–1) was purchased from DuPont. The standard incubation mixture (50 µl) contained 50 mM Tris–HCl pH 7.5, 10 mM MgCl2, 1.0 µM [γ-32P]GTP (60,000 dpm pmol–1) and 5 pmol of recombinant proteins. After incubation for 10 min at 25°C, the reaction was stopped by the addition of 450 µl of 5% (w/v) Norit in 50 mM NaH2PO4 (4°C). The charcoal was removed by centrifugation and the radioactivity in an aliquot of the supernatant was determined with a liquid scintillation spectrometer. Measurement of the activity of [γ-32P]GTP bound to recombinant proteins was carried out at 25°C using established procedures. The standard reaction mixture (100 µl) consisted of 50 mM Tris–HCl pH 7.5, 10 µM MgCl2, 1.0 µM [γ-32P]GTP (60,000 dpm pmol–1) and 10 pmol recombinant proteins. After incubation for 10 min, the reaction mixture was applied to a Sephadex G50 column (Boehringer Mannheim) equilibrated with 50 mM Tris–HCl pH 7.5 and 10 µM MgCl2 in order to remove unbound [γ-32P]GTP. The fractions containing [γ-32P]GTP-bound proteins were incubated further for the indicated times and filtered under vacuum through a 13 mm nitrocellulose membrane (pore size 0.45 µm, Millipore) to remove hydrized γ-32P. The membrane was washed five times with 300 µl of an ice-cold stop solution (20 mM Tris–HCl pH 8.0, 100 mM NaCl, 20 mM MgCl2). The radioactivity on the membrane was determined by a liquid scintillation spectrometer. A chimeric gene, ProRGA1:QL, was constructed as described previously (Kato et al. 2004). Briefly, a 2.7 kbp fragment, excised using SalI and KpnI, that included 1 kbp of the promoter region to the third exon of the RGA1 gene, was isolated and designated as the ProRGA1 fragment. A cDNA fragment, stretching from the first to third exons, was removed by SalI and KpnI digestion from pKF-QL and replaced with the ProRGA1 fragment; the resultant chimeric clone was designated ProRGA1:QL. The QL mutation was in the tenth exon. The binary vector pB101-Hm, expressing β-glucoronidase (GUS) under control of the cauliflower mosaic virus 35S promoter, was used as the control vector for rice transformation. Part of the 35S promoter and the GUS gene of pBI121-Hm were replaced with the chimeric clone, ProRGA1:QL. Transgenic rice plants were generated using the Agrobacterium-mediated transformation methods described previously (Toki 1997). Dehusked d1 seeds were sterilized and inoculated on the callus induction plates. After 3 weeks, calli proliferating from the scutella were used for transformation. EHA101, containing the binary vector, was co-cultured with rice d1 calli and transgenic rice plants were selected in the presence of hygromycin. The accumulation of Q223L protein was analyzed by Western blotting using an anti-α antibody. Transformants expressing Q223L protein were named QL/d1. Acknowledgments We thank Dr. Hikaru Satoh for the gift of DK22 and Dr. Tadashi Asahi for his critical review of this manuscript. Part of the work was carried out at the Biological Resource Research and Development Center, Fukui Prefectural University. We acknowledge funding from two sources: a Grant-in-Aid for Scientific Research on Priority Areas (no.15031223) from the Ministry of Education, Science and Culture, Japan to Y.I., and a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Functional analysis of genes relevant to agriculturally important traits in rice genome IP-1002) to Y.I. Open in new tabDownload slide Fig. 1 Alignment of the amino acid sequences of the α subunits of heterotrimeric G proteins from rice (RGA1), Arabidopsis thaliana (GPA1), human Gt and Gs and of the human small G protein (c-Ha-ras). Accession numbers are as follows: RGA1 (P49083), GPA1 (NP 180198), human Gt (1GOT A), human Gs (1AZT B) and c-Ha-ras (1Q21). Arrowheads (G48V, R191C, Q223L and A356S) indicate the mutation sites of RGA1. GTP-binding sites are shown as box A, box C, box G and box I. Effector-binding regions and a receptor-binding region are underlined. Arginine at position 191 is known to be a site ADP-ribosylated by cholera toxin. Open in new tabDownload slide Fig. 1 Alignment of the amino acid sequences of the α subunits of heterotrimeric G proteins from rice (RGA1), Arabidopsis thaliana (GPA1), human Gt and Gs and of the human small G protein (c-Ha-ras). Accession numbers are as follows: RGA1 (P49083), GPA1 (NP 180198), human Gt (1GOT A), human Gs (1AZT B) and c-Ha-ras (1Q21). Arrowheads (G48V, R191C, Q223L and A356S) indicate the mutation sites of RGA1. GTP-binding sites are shown as box A, box C, box G and box I. Effector-binding regions and a receptor-binding region are underlined. Arginine at position 191 is known to be a site ADP-ribosylated by cholera toxin. Open in new tabDownload slide Fig. 2 Purification of recombinant α subunits from the total lysates of E. coli, and GTPase and GTP-binding activities of recombinant proteins. (A) SDS–PAGE. rRGA1, rG48V, rR191C and rA356S were purified by Ni2+ affinity column chromatography and Mono Q column chromatography. A 5 µg aliquot of each purified recombinant protein was electrophoresed on a 12.5% polyacrylamide gel and proteins were visualized by staining with Coomassie brilliant blue R-250. Lane 1, molecular weight marker; lane 2, rRGA1; lane 3, rG48V; lane 4, rR191C; lane 5, rQ223L; lane 6, rA356S. (B) Time course analysis of GTPase hydrolysis by purified recombinant proteins. All recombinant proteins were incubated with [γ-32P]GTP at 25°C and the reaction was terminated at the indicated times. Released 32P was quantified with a liquid scintillation spectrometer. Data shown represent the means of duplicate measurements. rRGA1 (filled diamond), rG48V (open square), rR191C (filled square), rQ223L (filled triangle), rA356S (open circle). (C) [γ-32P]GTP-binding of purified recombinant proteins. The purified recombinant proteins were incubated with [γ-32P]GTP for 10 min, and then each of the incubation mixtures was applied to a Sephadex G 50 column to remove unbound [γ-32P]GTP from protein-bound [γ-32P]GTP. The protein fractions obtained in this way were incubated further with the reaction buffer and the reactions were terminated at the indicated times. rRGA1 (filled diamond), rG48V (open square), rR191C (filled square), rQ223L (closed triangle), rA356S (open circle). Open in new tabDownload slide Fig. 2 Purification of recombinant α subunits from the total lysates of E. coli, and GTPase and GTP-binding activities of recombinant proteins. (A) SDS–PAGE. rRGA1, rG48V, rR191C and rA356S were purified by Ni2+ affinity column chromatography and Mono Q column chromatography. A 5 µg aliquot of each purified recombinant protein was electrophoresed on a 12.5% polyacrylamide gel and proteins were visualized by staining with Coomassie brilliant blue R-250. Lane 1, molecular weight marker; lane 2, rRGA1; lane 3, rG48V; lane 4, rR191C; lane 5, rQ223L; lane 6, rA356S. (B) Time course analysis of GTPase hydrolysis by purified recombinant proteins. All recombinant proteins were incubated with [γ-32P]GTP at 25°C and the reaction was terminated at the indicated times. Released 32P was quantified with a liquid scintillation spectrometer. Data shown represent the means of duplicate measurements. rRGA1 (filled diamond), rG48V (open square), rR191C (filled square), rQ223L (filled triangle), rA356S (open circle). (C) [γ-32P]GTP-binding of purified recombinant proteins. The purified recombinant proteins were incubated with [γ-32P]GTP for 10 min, and then each of the incubation mixtures was applied to a Sephadex G 50 column to remove unbound [γ-32P]GTP from protein-bound [γ-32P]GTP. The protein fractions obtained in this way were incubated further with the reaction buffer and the reactions were terminated at the indicated times. rRGA1 (filled diamond), rG48V (open square), rR191C (filled square), rQ223L (closed triangle), rA356S (open circle). Open in new tabDownload slide Fig. 3 Transgenic rice plants expressing Q223L. (A) Schematic diagrams of the structures of the chimeric gene, ProRGA1:QL. The C-terminal part of mutagenized Q223L cDNA, which included the site of animo acid exchange, was fused to the RGA1 gene at the third exon. (B) Photograph of rice plants after flowering. A chimeric gene, ProRGA1:QL, was introduced into d1, a rice mutant defective in the α-subunit gene, by the Agrobacterium-mediated transformation method. d1, a rice mutant defective in the α-subunit gene; NC, normal cultivar; QL/d1, transformants with a chimeric gene, ProRGA1:QL, introduced into d1. (C) Western blot analysis. Plasma membrane proteins from d1, NC and QL/d1 were separated by SDS–PAGE and identified on Western blots using anti-α antibody. The position of the RGA1 and Q223L proteins is indicated by an arrow. (D) Photograph of rice seed of NC, d1 and QL/d1. Unhulled and hulled seeds are shown in the upper and lower parts, respectively. Open in new tabDownload slide Fig. 3 Transgenic rice plants expressing Q223L. (A) Schematic diagrams of the structures of the chimeric gene, ProRGA1:QL. The C-terminal part of mutagenized Q223L cDNA, which included the site of animo acid exchange, was fused to the RGA1 gene at the third exon. (B) Photograph of rice plants after flowering. A chimeric gene, ProRGA1:QL, was introduced into d1, a rice mutant defective in the α-subunit gene, by the Agrobacterium-mediated transformation method. d1, a rice mutant defective in the α-subunit gene; NC, normal cultivar; QL/d1, transformants with a chimeric gene, ProRGA1:QL, introduced into d1. (C) Western blot analysis. Plasma membrane proteins from d1, NC and QL/d1 were separated by SDS–PAGE and identified on Western blots using anti-α antibody. The position of the RGA1 and Q223L proteins is indicated by an arrow. (D) Photograph of rice seed of NC, d1 and QL/d1. Unhulled and hulled seeds are shown in the upper and lower parts, respectively. Table 1 Length and width of unhulled rice seeds Normal cultivar d1 QL/d1 10 27 33 43 46 Length 7.59 ± 0.44 4.79 ± 0.43 7.78 ± 0.36 7.98 ± 0.47 7.88 ± 0.41 7.77 ± 0.34 8.01 ± 0.36 Width 3.68 ± 0.35 3.54 ± 0.36 3.65 ± 0.34 3.72 ± 0.37 3.86 ± 0.33 3.51 ± 0.32 3.79 ± 0.36 Normal cultivar d1 QL/d1 10 27 33 43 46 Length 7.59 ± 0.44 4.79 ± 0.43 7.78 ± 0.36 7.98 ± 0.47 7.88 ± 0.41 7.77 ± 0.34 8.01 ± 0.36 Width 3.68 ± 0.35 3.54 ± 0.36 3.65 ± 0.34 3.72 ± 0.37 3.86 ± 0.33 3.51 ± 0.32 3.79 ± 0.36 Length and width are shown in mm. Data are the averages of 10 plants (±SD). Open in new tab Table 1 Length and width of unhulled rice seeds Normal cultivar d1 QL/d1 10 27 33 43 46 Length 7.59 ± 0.44 4.79 ± 0.43 7.78 ± 0.36 7.98 ± 0.47 7.88 ± 0.41 7.77 ± 0.34 8.01 ± 0.36 Width 3.68 ± 0.35 3.54 ± 0.36 3.65 ± 0.34 3.72 ± 0.37 3.86 ± 0.33 3.51 ± 0.32 3.79 ± 0.36 Normal cultivar d1 QL/d1 10 27 33 43 46 Length 7.59 ± 0.44 4.79 ± 0.43 7.78 ± 0.36 7.98 ± 0.47 7.88 ± 0.41 7.77 ± 0.34 8.01 ± 0.36 Width 3.68 ± 0.35 3.54 ± 0.36 3.65 ± 0.34 3.72 ± 0.37 3.86 ± 0.33 3.51 ± 0.32 3.79 ± 0.36 Length and width are shown in mm. Data are the averages of 10 plants (±SD). Open in new tab Table 2 Weight of rice seeds Normal cultivar d1 QL/d1 10 27 33 43 46 Unhulled seed 26.0 ± 1.03 14.8 ± 0.87 29.4 ± 1.26 30.4 ± 1.16 30.1 ± 1.09 29.3 ± 1.54 30.6 ± 1.47 Hull 4.50 ± 0.41 3.50 ± 0.49 4.70 ± 0.52 5.41 ± 0.43 5.22 ± 0.61 5.01 ± 0.52 5.92 ± 0.41 Hulled seed 21.5 ± 1.04 11.3 ± 0.81 24.7 ± 1.23 25.0 ± 1.18 24.9 ± 1.11 24.3 ± 1.42 24.7 ± 1.39 Normal cultivar d1 QL/d1 10 27 33 43 46 Unhulled seed 26.0 ± 1.03 14.8 ± 0.87 29.4 ± 1.26 30.4 ± 1.16 30.1 ± 1.09 29.3 ± 1.54 30.6 ± 1.47 Hull 4.50 ± 0.41 3.50 ± 0.49 4.70 ± 0.52 5.41 ± 0.43 5.22 ± 0.61 5.01 ± 0.52 5.92 ± 0.41 Hulled seed 21.5 ± 1.04 11.3 ± 0.81 24.7 ± 1.23 25.0 ± 1.18 24.9 ± 1.11 24.3 ± 1.42 24.7 ± 1.39 Weight is shown in mg. Data are the averages of 10 plants (±SD). Open in new tab Table 2 Weight of rice seeds Normal cultivar d1 QL/d1 10 27 33 43 46 Unhulled seed 26.0 ± 1.03 14.8 ± 0.87 29.4 ± 1.26 30.4 ± 1.16 30.1 ± 1.09 29.3 ± 1.54 30.6 ± 1.47 Hull 4.50 ± 0.41 3.50 ± 0.49 4.70 ± 0.52 5.41 ± 0.43 5.22 ± 0.61 5.01 ± 0.52 5.92 ± 0.41 Hulled seed 21.5 ± 1.04 11.3 ± 0.81 24.7 ± 1.23 25.0 ± 1.18 24.9 ± 1.11 24.3 ± 1.42 24.7 ± 1.39 Normal cultivar d1 QL/d1 10 27 33 43 46 Unhulled seed 26.0 ± 1.03 14.8 ± 0.87 29.4 ± 1.26 30.4 ± 1.16 30.1 ± 1.09 29.3 ± 1.54 30.6 ± 1.47 Hull 4.50 ± 0.41 3.50 ± 0.49 4.70 ± 0.52 5.41 ± 0.43 5.22 ± 0.61 5.01 ± 0.52 5.92 ± 0.41 Hulled seed 21.5 ± 1.04 11.3 ± 0.81 24.7 ± 1.23 25.0 ± 1.18 24.9 ± 1.11 24.3 ± 1.42 24.7 ± 1.39 Weight is shown in mg. Data are the averages of 10 plants (±SD). Open in new tab Table 3 Length of ear and internodes of rice plants after flowering Normal cultivar d1 QL/d1 10 27 33 43 46 Ear 19.7 ± 1.8 13.1 ± 1.1 18.8 ± 1.8 20.6 ± 1.9 20.4 ± 1.6 18.6 ± 1.2 20.9 ± 1.6 First internode 32.6 ± 3.7 17.9 ± 1.4 34.8 ± 1.8 35.8 ± 3.6 36.4 ± 1.9 33.7 ± 2.1 35.2 ± 2.5 Second internode 16.3 ± 1.9 6.5 ± 0.5 16.7 ± 1.9 17.5 ± 1.5 17.3 ± 1.0 16.8 ± 1.1 18.2 ± 1.4 Third internode 8.9 ± 1.8 3.0 ± 1.0 8.3 ± 1.4 7.6 ± 1.6 7.7 ± 0.9 8.5 ± 1.6 8.8 ± 2.3 Fourth internode 4.0 ± 1.9 0.8 ± 0.5 3.0 ± 1.4 3.2 ± 1.7 3.5 ± 1.8 4.6 ± 2.0 3.6 ± 2.5 Total 82.0 ± 6.8 41.3 ± 2.2 82.2 ± 5.1 85.3 ± 7.6 86.0 ± 4.9 83.0 ± 4.2 87.3 ± 4.6 Normal cultivar d1 QL/d1 10 27 33 43 46 Ear 19.7 ± 1.8 13.1 ± 1.1 18.8 ± 1.8 20.6 ± 1.9 20.4 ± 1.6 18.6 ± 1.2 20.9 ± 1.6 First internode 32.6 ± 3.7 17.9 ± 1.4 34.8 ± 1.8 35.8 ± 3.6 36.4 ± 1.9 33.7 ± 2.1 35.2 ± 2.5 Second internode 16.3 ± 1.9 6.5 ± 0.5 16.7 ± 1.9 17.5 ± 1.5 17.3 ± 1.0 16.8 ± 1.1 18.2 ± 1.4 Third internode 8.9 ± 1.8 3.0 ± 1.0 8.3 ± 1.4 7.6 ± 1.6 7.7 ± 0.9 8.5 ± 1.6 8.8 ± 2.3 Fourth internode 4.0 ± 1.9 0.8 ± 0.5 3.0 ± 1.4 3.2 ± 1.7 3.5 ± 1.8 4.6 ± 2.0 3.6 ± 2.5 Total 82.0 ± 6.8 41.3 ± 2.2 82.2 ± 5.1 85.3 ± 7.6 86.0 ± 4.9 83.0 ± 4.2 87.3 ± 4.6 The length is shown in cm. Data are the averages of 10 plants (±SD). Open in new tab Table 3 Length of ear and internodes of rice plants after flowering Normal cultivar d1 QL/d1 10 27 33 43 46 Ear 19.7 ± 1.8 13.1 ± 1.1 18.8 ± 1.8 20.6 ± 1.9 20.4 ± 1.6 18.6 ± 1.2 20.9 ± 1.6 First internode 32.6 ± 3.7 17.9 ± 1.4 34.8 ± 1.8 35.8 ± 3.6 36.4 ± 1.9 33.7 ± 2.1 35.2 ± 2.5 Second internode 16.3 ± 1.9 6.5 ± 0.5 16.7 ± 1.9 17.5 ± 1.5 17.3 ± 1.0 16.8 ± 1.1 18.2 ± 1.4 Third internode 8.9 ± 1.8 3.0 ± 1.0 8.3 ± 1.4 7.6 ± 1.6 7.7 ± 0.9 8.5 ± 1.6 8.8 ± 2.3 Fourth internode 4.0 ± 1.9 0.8 ± 0.5 3.0 ± 1.4 3.2 ± 1.7 3.5 ± 1.8 4.6 ± 2.0 3.6 ± 2.5 Total 82.0 ± 6.8 41.3 ± 2.2 82.2 ± 5.1 85.3 ± 7.6 86.0 ± 4.9 83.0 ± 4.2 87.3 ± 4.6 Normal cultivar d1 QL/d1 10 27 33 43 46 Ear 19.7 ± 1.8 13.1 ± 1.1 18.8 ± 1.8 20.6 ± 1.9 20.4 ± 1.6 18.6 ± 1.2 20.9 ± 1.6 First internode 32.6 ± 3.7 17.9 ± 1.4 34.8 ± 1.8 35.8 ± 3.6 36.4 ± 1.9 33.7 ± 2.1 35.2 ± 2.5 Second internode 16.3 ± 1.9 6.5 ± 0.5 16.7 ± 1.9 17.5 ± 1.5 17.3 ± 1.0 16.8 ± 1.1 18.2 ± 1.4 Third internode 8.9 ± 1.8 3.0 ± 1.0 8.3 ± 1.4 7.6 ± 1.6 7.7 ± 0.9 8.5 ± 1.6 8.8 ± 2.3 Fourth internode 4.0 ± 1.9 0.8 ± 0.5 3.0 ± 1.4 3.2 ± 1.7 3.5 ± 1.8 4.6 ± 2.0 3.6 ± 2.5 Total 82.0 ± 6.8 41.3 ± 2.2 82.2 ± 5.1 85.3 ± 7.6 86.0 ± 4.9 83.0 ± 4.2 87.3 ± 4.6 The length is shown in cm. Data are the averages of 10 plants (±SD). Open in new tab Abbreviations GA gibberellin GUS β-glucuronidase References Aharon, G.S., Gelli, A., Snedden, W.A. and Blumwald, E. ( 1998 ) Activation of a plant plasma membrane Ca2+ channel by TGα1, a heterotrimeric G protein α-subunit homologue. FEBS Lett. 424 : 17 –21. Ashikari, M., Wu, J., Yano, M., Sasaki, T. and Yoshimura, A. ( 1999 ) Rice gibberellin-insensitive dwarf mutant gene Dwarf 1 encodes the α-subunit of GTP-binding protein. Proc. Natl Acad. Sci. USA 96 : 10284 –10289. Chen, J.G., Willard, F.S., Huang, J., Liang, J., Chasse, S.A., Jones, A.M. and Siderovski, D.P. ( 2003 ) A seven-transmembrane RGS protein that modulates plant cell proliferation. Science 301 : 1728 –1731. Clapham, D.E. and Neer, E.J. ( 1993 ) New roles for G-protein βγ-dimers in transmembrane signalling. Nature 365 : 403 –406. Coursol, S., Fan, L.M., Le Stunff, H., Spiegel, S., Gilroy, S. and Assmann, S.M. ( 2003 ) Sphingolipid signalling in Arabidopsis guard cells involves heterotrimeric G proteins. Nature 423 : 651 –654. Ellis, B.E. and Miles, G.P. ( 2001 ) Plant biology. One for all? Science 292 : 2022 –2023. Fujisawa, Y., Kato, H. and Iwasaki, Y. ( 2001 ) Structure and function of heterotrimeric G proteins in plants. Plant Cell Physiol. 42 : 789 –794. Fujisawa, Y., Kato, T., Ohki, S., Ishikawa, A., Kitano, H., Sasaki, T., Asahi, T. and Iwasaki, Y. ( 1999 ) Suppression of the heterotrimeric G protein causes abnormal morphology, including dwarfism, in rice. Proc. Natl Acad. Sci. USA 96 : 7575 –7580. Fujisawa, Y., Sawaki, S., Kato, H., Asahi, T. and Iwasaki, Y. ( 2001 ) Biochemical responses of rice cells to mastoparan 7, an activator of heterotrimeric G proteins. Plant Biotechnol. 18 : 241 –249. Higashijima, T., Ferguson, K.M., Smigel, M.D. and Gilman, A.G. ( 1987 ) The effect of GTP and Mg2+ on the GTPase activity and the fluorescent properties of Go. J. Biol. Chem. 262 : 757 –761. Iwasaki, Y., Fujisawa, Y. and Kato, H. ( 2002 ) Function of heterotrimeric G protein in gibberellin signaling. J. Plant Growth Regul. 22 : 126 –133. Iwasaki, Y., Kato, T., Kaidoh, T., Ishikawa, A. and Asahi, T. ( 1997 ) Characterization of the putative α subunit of a heterotrimeric G protein in rice. Plant Mol. Biol. 34 : 563 –572. Jones, A.M. and Assmann, S.M. ( 2004 ) Plants: the latest model system for G-protein research. EMBO Rep. 5 : 572 –578. Jones, H.D., Smith, S.J., Desikan, R., Plakidou-Dymock, S., Lovegrove, A. and Hooley, R. ( 1998 ) Heterotrimeric G proteins are implicated in gibberellin induction of α-amylase gene expression in wild oat aleurone. Plant Cell 10 : 245 –254. Kato, C., Mizutani, T., Tamaki, H., Kumagai, H., Kamiya, T., Hirobe, A., Fujisawa, Y., Kato, H. and Iwasaki, Y. ( 2004 ) Characterization of heterotrimeric G protein complexes in rice plasma membrane. Plant J. 38 : 320 –331. Kaziro, Y., Itoh, H., Kozasa, T., Nakafuku, M. and Satoh, T. ( 1991 ) Structure and function of signal-transducing GTP-binding proteins. Annu. Rev. Biochem. 60 : 349 –400. Lapik, Y.R. and Kaufman, L.S. ( 2003 ) The Arabidopsis cupin domain protein AtPirin1 interacts with the G protein α-subunit GPA1 and regulates seed germination and early seedling. development. Plant Cell 15 : 1578 –1590. Lease, K.A., Wen, J., Li, J., Doke, J.T., Liscum, E. and Walker, J.C. ( 2001 ) A mutant Arabidopsis heterotrimeric G-protein β subunit affects leaf, flower, and fruit development. Plant Cell 13 : 2631 –2641. Lovegrove, A. and Hooley, R. ( 2000 ) Gibberellin and abscisic acid signalling in aleurone. Trends Plant Sci. 5 : 102 –110. Mitsunaga, S., Tashiro, T. and Yamaguchi, J. ( 1994 ) Identification and characterization of gibberellin-insensitive mutants selected from among dwarf mutants of rice. Theor. Appl. Genet. 87 : 705 –712. Okamoto, H., Matsui, M. and Deng, X.W. ( 2001 ) Overexpression of the heterotrimeric G-protein α-subunit enhances phytochrome-mediated inhibition of hypocotyl elongation in Arabidopsis. Plant Cell 13 : 1639 –1652. Pandey, S. and Assmann, S.M. ( 2004 ) The Arabidopsis putative G protein-coupled receptor GCR1 interacts with the G protein α subunit GPA1 and regulates abscisic acid signaling. Plant Cell 16 : 1616 –1632. Ross, E.M. and Higashijima, T. ( 1994 ) Regulation of G-protein activation by mastoparans and other cationic peptides. Methods Enzymol. 237 : 26 –37. Suharsono, U., Fujisawa, Y., Kawasaki, T., Iwasaki, Y., Satoh, H. and Shimamoto, K. ( 2002 ) The heterotrimeric G protein α subunit acts upstream of the small GTPase Rac in disease resistance of rice. Proc. Natl Acad. Sci. USA 99 : 13307 –13312. Toki, S. ( 1997 ) Rapid and efficient Agrobacterium-mediated transformation in rice. Plant Mol. Biol. Rep. 15 : 16 –21. Ueguchi-Tanaka, M., Fujisawa, Y., Kobayashi, M., Ashikari, M., Iwasaki, Y., Kitano, H. and Matsuoka, M. ( 2000 ) Rice dwarf mutant d1, which is defective in the α subunit of the heterotrimeric G protein, affects gibberellin signal transduction. Proc. Natl Acad. Sci. USA 97 : 11638 –11643. Ullah, H., Chen, J.G., Young, J.C., Im, K.H., Sussman, M.R. and Jones, A.M. ( 2001 ) Modulation of cell proliferation by heterotrimeric G protein in Arabidopsis. Science 292 : 2066 –2069. Zhao, J. and Wang, X. ( 2004 ) Arabidopsis phospholipase Dα1 interacts with the heterotrimeric G-protein α-subunit through a motif analogous to the DRY motif in G-protein-coupled receptors. J. Biol. Chem. 279 : 1794 –1800. JSPP © 2005 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

Study of the Constitutively Active Form of the α Subunit of Rice Heterotrimeric G Proteins

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Oxford University Press
Copyright
JSPP © 2005
ISSN
0032-0781
eISSN
1471-9053
DOI
10.1093/pcp/pci036
pmid
15695461
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Abstract

Abstract We used site-directed mutagenesis to engineer two constitutively active forms of the α subunit of a rice heterotrimeric G protein. The recombinant proteins produced from these novel cDNAs had GTP-binding activity but no GTPase activity. A chimeric gene for a constitutively active form of the α subunit was introduced into the rice mutant d1, which is defective for the α-subunit gene. All the transformants essentially showed a wild-type phenotype compared with normal cultivars, although seed sizes were substantially increased and internode lengths also showed some increase. Heterotrimeric G proteins regulate the activity of downstream effectors following perception of external signals by receptors. Agonist-induced stimulation of G protein-coupled receptors causes heterotrimeric G proteins to dissociate into α subunits and βγ dimer subunits. In mammals, dissociated α subunits and βγ dimers transmit the receptor-generated signals to effectors in an antagonistic, synergistic or independent manner, depending upon the species of the three subunits (Clapham and Neer 1993). Progress in our understanding of the function of plant heterotrimeric G proteins has been advanced through analysis of mutants that have defects in the subunit genes (Ashikari et al. 1999, Fujisawa et al. 1999, Lease et al. 2001, Ullah et al. 2001). Mutants of the α-subunit gene isolated from rice and Arabidopsis were designated as d1 (Ashikari et al. 1999, Fujisawa et al. 1999) and gpa1 (Ullah et al. 2001), respectively. Studies on d1 have suggested the G proteins may be involved in more than three signaling pathways, including the gibberellin (GA) (Ueguchi-Tanaka et al. 2000), pathogen infection response (Suharsono et al. 2002) and light (Iwasaki et al. 2002) signaling pathways. d1 shows abnormal morphology such as dwarfism, dark green leaves and small round seed. The findings revealed that the G proteins are functional molecules regulating the body plans in rice. Studies on gpa1 raised the possibility that the G protein may be involved a number of signaling pathways, including those of GA, auxin, abscisic acid, ethylene, light and sugar (Ellis and Miles 2001, Ullah et al. 2001). Recently, it was proposed that sphingosine-1-phosphate is one of the signal molecules of the G protein-mediated signaling pathways in plants (Coursol et al. 2003). In addition, a putative G protein-coupled receptor (GCR1) (Pandey and Assmann 2004) and a regulator of G protein signaling (RGS) (Chen et al. 2003) have been shown to interact with the α subunits of heterotrimeric G proteins. Various proteins, for example cupin domain (Lapik and Kaufman 2003), calcium channel (Aharon et al. 1998) and a phospholipase D (Zhao and Wang 2004), have been proposed as effectors targeted by G proteins in plants. There is, however, little definitive information on the connection or relationship between putative signals, receptors and effectors in plants. In mammals, activators of G proteins, such as mastoparan, cholera toxin and GTPγS, and inhibitors of G proteins, such as GDPβS and pertussis toxin, are useful tools in the analysis of G protein-mediated signaling (Kaziro et al. 1991). However, mastoparan affects not only the G protein but also other proteins such as calmodulin and phospholipase C in mammals (Ross and Higashijima 1994). In addition, mastoparan 7, an active analog of mastoparan, was found to cause damage to cells in a G protein-independent manner in rice plants (Fujisawa et al. 2001b). Much controversy has arisen over whether the use of these activators and inhibitors is appropriate for the study of the G protein-mediated signaling in plants (Fujisawa et al. 2001a). An alternative approach that avoids such controversy is to convert the α subunits to constitutively active forms by amino acid substitution. Constitutively active α subunits regulate downstream effectors even in the absence of signals and stimulated receptors. As a result, constitutively active subunits are valuable tools for investigating G protein-mediated signaling in plants. In this report, we describe formation of constitutively active forms of the α subunit of rice heterotrimeric G proteins as a result of amino acid substitutions mediated by site-directed mutagenesis. We show that the constitutively active forms are able to rescue the abnormal phenotype of rice mutants defective in the α-subunit gene. It has been shown that exchange of only one amino acid in the sequence of α subunits of mammalian heterotrimeric G proteins is sufficient to result in a constitutively active form (Kaziro et al. 1991). On this basis, we introduced mutations into the α subunit (RGA1) of rice (Fig. 1). In one example, site-directed mutagenesis of RGA1 cDNA at position 223 caused the substitution of leucine (L) by glutamine (Q); the resultant clone was designated as Q223L cDNA. We introduced four mutations into RGA1 cDNA each with a different amino acid substitution at a different position, clones G48V, R191C, Q223L and A356S cDNAs (Fig. 1). Previously, we showed that a recombinant α subunit (RGA1) could be synthesized in a soluble form in Escherichia coli and purified (Iwasaki et al. 1997). In the present work, the site-directed mutagenized recombinant proteins (rG48V, rR191C, rQ223L and rA356S) were synthesized in E. coli and purified, according to the same procedures. All recombinant proteins were highly purified in water-soluble form, not as inclusion bodies in E. coli (Fig. 2A). When an α subunit is forced to become constitutively active through amino acid exchange, its GTPase and GTP-binding activities may be altered. Therefore, we first screened the GTPase activity of purified rRGA1, rG48V, rR191C, rQ223L and rA356S (Fig. 2B). Two of these, rQ223L and rR191C, had lost all GTPase activity, whereas the other two, rG48V and rA356S, still showed some GTPase activity albeit at much lower levels than in rRGA1. Next, we assayed the GTP-binding activity of the recombinant proteins (Fig. 2C). The proteins were incubated with [γ-32P]GTP and the amount of bound [γ-32P]GTP measured by liquid scintillation spectrometry. All four recombinant proteins were found to bind [γ-32P]GTP; however, they differed in their abilities to retain [γ-32P]GTP at longer incubation times. The [γ-32P]GTP-bound rQ223L and rR191C retained [γ-32P]GTP during further incubation. The [γ-32P]GTP-bound rG48V and rA356S lost γ-32P with time; they did not lose GTPase activity (Fig. 2C). Thus, rQ223L and rR191C keep the ability to bind GTP and lose GTPase activity. The results strongly indicate that rQ223L and rR191C are constitutively active forms of the α subunit. In vitro mutagenized α subunits of plant heterotrimeric G proteins have also been constructed for Arabidopsis (Okamoto et al. 2001) and tomato (Aharon et al. 1998). No biochemical data were reported for the Arabidopsis protein corresponding to Q223L of rice. The tomato protein corresponding to Q223L was fused to glutathione S-transferase; it appeared to have no GTPase activity but to have GTP-binding activity. However, no kinetic data were presented (principally because GTP binding was measured on the membrane after SDS–PAGE), and thus further analysis is necessary to confirm that the tomato protein truly lacks GTPase activity but has GTP-binding activity. Our use of purified proteins and detailed enzyme characterization has enabled us to conclude that both R191C and Q223L are constitutively active forms of the α subunit of the rice heterotrimeric G proteins. Part of the cDNA for Q223L was fused to the RGA1 gene to construct a chimeric gene, ProRGA1:QL (Fig. 3A). ProRGA1:QL is controlled by the promoter of the RGA1 gene and contains two introns in the RGA1 gene fragment. After the chimeric gene was fused to a binary vector, pBI-121Hm, it was introduced into calli of the rice mutant, d1, via the Agrobacterium transformation method. The dl mutant is defective in the gene of the α subunit of heterotrimeric G proteins and thus contains no α subunit. Hygromycin-resistant transformants were selected for Q223L expression by reverse transcription–polymerase chain reaction (RT–PCR). Mutant d1 plants show dwarfism, bear small round seeds, and form dark green leaves. In contrast, all rice transformants accumulating Q223L protein on the d1 background showed an essentially wild-type phenotype (Fig. 3B). The level of Q223L protein in the transformants was assayed using an anti-α subunit antibody. A typical Western blot, from the QL/d1 line of selected transformants, is shown in Fig. 3C. The constitutively active form of the α subunit accumulated in QL/d1 at the same level as seen in the normal cultivar. The unhulled seeds of transformants were about 5% longer than those of a normal cultivar, although unhulled seed widths were similar (Fig. 3D, Table 1). In transformants, unhulled seeds, hulled seeds and the hulls were approximately 20% heavier than those of normal cultivar rice (Table 2). Internode lengths of QL/d1 were about 5% longer than those of the normal cultivar (Table 3). Our data clearly show that constitutive activation of the α subunit by Q223L had a more substantial effect on seed formation than on internode length. Plant heterotrimeric G proteins are involved in multiple signaling pathways, including light, plant hormones and elicitor, as demonstrated by comparisons of signal responses of wild-type plants and G protein subunit mutants (Ellis and Miles 2001, Iwasaki et al. 2002, Jones and Assmann 2004). Transformants that have constitutive overexpression of the α subunit have been studied in Arabidopsis, oats and rice with the purpose of further elucidating the signal pathways through which heterotrimeric G proteins act. A study of transformants overexpressing the constitutively active α subunit (Q222L) in Arabidopsis suggested that the α subunit of heterotrimeric G proteins may act on a discrete branch of the phytochrome A signaling pathway; the α subunit seemed to be involved indirectly rather than directly with light signaling (Okamoto et al. 2001). In oats (Jones et al. 1998) and rice (Mitsunaga et al. 1994, Ueguchi-Tanaka et al. 2000), before discovery of G protein mutants, it was believed initially that the heterotrimeric G proteins were concerned with GA signaling. The identification of the rice dwarf mutant, d1, which has a mutation in the α subunit gene of the heterotrimeric G proteins, supported this perception. The mRNA level and enzymatic activity of α-amylase after application of GA3 were compared in embryo-less half-seeds of d1 and the normal cultivar. The d1 mutant gave a reduced response to low concentrations of GA3 compared with the normal cultivar, but a similar response at high concentration (Ueguchi-Tanaka et al. 2000). From these results, we considered that rice heterotrimeric G proteins were likely to be involved with GA signaling at low, but not high, concentrations of GAs (Iwasaki et al. 2002). There is no unequivocal evidence that G proteins directly regulate the GA signaling pathway through the transcriptional activation of GAMyb, a positive transcriptional factor of the α-amylase gene. It cannot be ruled out that the G proteins are indirect regulators of the GA signaling pathway. Lovegrove and Hooley (2000) proposed that such indirect regulation could arise through post-translational events after the perception of GAs. In the present work, rice dl transformants expressing Q223L did not show a slender phenotype but rather were essentially wild type (Fig. 3B). This suggests that it is unlikely that rice heterotrimeric G proteins are concerned with the major GA signaling pathways. Embryo-less half-seeds need GA for synthesis of α-amylase. If the constitutively active form of the α subunit was a positive regulator for major GA signaling, then it would be expected that α-amylase would be induced in embryo-less half-seeds of QL/d1 without GA. However, when we measured the activity of α-amylase in the embryo-less half-seeds of the transformants and normal cultivars (Mitsunaga et al. 1994), we found that the α-amylase content of QL/d1 was similar to that of the normal cultivar (data not shown). Consequently, we believe that heterotrimeric G proteins do not directly regulate the GA signaling pathway in rice. The present work shows that Q223L plays important roles in seed formation and thus we propose that Q223L would be a valuable tool to identify the G protein-mediated signaling pathways involved in seed formation. A rice normal cultivar (Oryza sativa L. cv. Nipponbare) and a rice dwarf mutant, d1 (DK22), were used in this study. The recurrent parent of d1 (DK22) was Nipponbare. All rice plants (normal cultivar, d1 and rice transformants) were grown under 14 h light, with cool white fluorescent light at 30,000 lux at 30°C, and 10 h dark at 25°C cycles. Site-directed mutagenesis was carried out using the Mutan-Express Km kit (Takara Bio Inc., Tokyo, Japan) in accordance with the manufacturer’s recommended protocol. The first step was to subclone the full-length sequence of RGA1 cDNA (GeneBank accession no. D38232) into the pKF19 plasmid. Four oligonucleotides were used in the site-directed mutagenesis experiments: G48V oligonucleotide (5′-TCCCTGATTCTACCGCACCAAG-3′); R191C oligonucleotide (5′-TTGTCCGTACACATGCATAAAGCAC-3′); Q223L oligonucleotide (5′-CTCATTCCTCAGGCCTCCTAC-3′); and A356S oligonucleotide (5′-CTGGTCTAGGCTCGTAGTTCTG-3′). All oligonucleotide sequences are shown in the antisense orientation of the RGA1 cDNA. The mutagenized constructs were sequenced to confirm mutagenesis and fidelity. The open reading frames of RGA1 and mutagenized relatives were subcloned, in-frame, in pQE30 vector (Qiagen K.K., Tokyo, Japan) and the pQE30 derivatives were transformed into E. coli BL21/DE3 with pREP4 plasmid. Recombinant proteins were synthesized in E. coli BL21/DE3 as described previously (Iwasaki et al. 1997). GTPase activity was assayed by the method, with minor modifications, described previously (Higashijima et al. 1987). [γ-32P]GTP (NEG-004Z, specific activity 222 TBq mmol–1) was purchased from DuPont. The standard incubation mixture (50 µl) contained 50 mM Tris–HCl pH 7.5, 10 mM MgCl2, 1.0 µM [γ-32P]GTP (60,000 dpm pmol–1) and 5 pmol of recombinant proteins. After incubation for 10 min at 25°C, the reaction was stopped by the addition of 450 µl of 5% (w/v) Norit in 50 mM NaH2PO4 (4°C). The charcoal was removed by centrifugation and the radioactivity in an aliquot of the supernatant was determined with a liquid scintillation spectrometer. Measurement of the activity of [γ-32P]GTP bound to recombinant proteins was carried out at 25°C using established procedures. The standard reaction mixture (100 µl) consisted of 50 mM Tris–HCl pH 7.5, 10 µM MgCl2, 1.0 µM [γ-32P]GTP (60,000 dpm pmol–1) and 10 pmol recombinant proteins. After incubation for 10 min, the reaction mixture was applied to a Sephadex G50 column (Boehringer Mannheim) equilibrated with 50 mM Tris–HCl pH 7.5 and 10 µM MgCl2 in order to remove unbound [γ-32P]GTP. The fractions containing [γ-32P]GTP-bound proteins were incubated further for the indicated times and filtered under vacuum through a 13 mm nitrocellulose membrane (pore size 0.45 µm, Millipore) to remove hydrized γ-32P. The membrane was washed five times with 300 µl of an ice-cold stop solution (20 mM Tris–HCl pH 8.0, 100 mM NaCl, 20 mM MgCl2). The radioactivity on the membrane was determined by a liquid scintillation spectrometer. A chimeric gene, ProRGA1:QL, was constructed as described previously (Kato et al. 2004). Briefly, a 2.7 kbp fragment, excised using SalI and KpnI, that included 1 kbp of the promoter region to the third exon of the RGA1 gene, was isolated and designated as the ProRGA1 fragment. A cDNA fragment, stretching from the first to third exons, was removed by SalI and KpnI digestion from pKF-QL and replaced with the ProRGA1 fragment; the resultant chimeric clone was designated ProRGA1:QL. The QL mutation was in the tenth exon. The binary vector pB101-Hm, expressing β-glucoronidase (GUS) under control of the cauliflower mosaic virus 35S promoter, was used as the control vector for rice transformation. Part of the 35S promoter and the GUS gene of pBI121-Hm were replaced with the chimeric clone, ProRGA1:QL. Transgenic rice plants were generated using the Agrobacterium-mediated transformation methods described previously (Toki 1997). Dehusked d1 seeds were sterilized and inoculated on the callus induction plates. After 3 weeks, calli proliferating from the scutella were used for transformation. EHA101, containing the binary vector, was co-cultured with rice d1 calli and transgenic rice plants were selected in the presence of hygromycin. The accumulation of Q223L protein was analyzed by Western blotting using an anti-α antibody. Transformants expressing Q223L protein were named QL/d1. Acknowledgments We thank Dr. Hikaru Satoh for the gift of DK22 and Dr. Tadashi Asahi for his critical review of this manuscript. Part of the work was carried out at the Biological Resource Research and Development Center, Fukui Prefectural University. We acknowledge funding from two sources: a Grant-in-Aid for Scientific Research on Priority Areas (no.15031223) from the Ministry of Education, Science and Culture, Japan to Y.I., and a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Functional analysis of genes relevant to agriculturally important traits in rice genome IP-1002) to Y.I. Open in new tabDownload slide Fig. 1 Alignment of the amino acid sequences of the α subunits of heterotrimeric G proteins from rice (RGA1), Arabidopsis thaliana (GPA1), human Gt and Gs and of the human small G protein (c-Ha-ras). Accession numbers are as follows: RGA1 (P49083), GPA1 (NP 180198), human Gt (1GOT A), human Gs (1AZT B) and c-Ha-ras (1Q21). Arrowheads (G48V, R191C, Q223L and A356S) indicate the mutation sites of RGA1. GTP-binding sites are shown as box A, box C, box G and box I. Effector-binding regions and a receptor-binding region are underlined. Arginine at position 191 is known to be a site ADP-ribosylated by cholera toxin. Open in new tabDownload slide Fig. 1 Alignment of the amino acid sequences of the α subunits of heterotrimeric G proteins from rice (RGA1), Arabidopsis thaliana (GPA1), human Gt and Gs and of the human small G protein (c-Ha-ras). Accession numbers are as follows: RGA1 (P49083), GPA1 (NP 180198), human Gt (1GOT A), human Gs (1AZT B) and c-Ha-ras (1Q21). Arrowheads (G48V, R191C, Q223L and A356S) indicate the mutation sites of RGA1. GTP-binding sites are shown as box A, box C, box G and box I. Effector-binding regions and a receptor-binding region are underlined. Arginine at position 191 is known to be a site ADP-ribosylated by cholera toxin. Open in new tabDownload slide Fig. 2 Purification of recombinant α subunits from the total lysates of E. coli, and GTPase and GTP-binding activities of recombinant proteins. (A) SDS–PAGE. rRGA1, rG48V, rR191C and rA356S were purified by Ni2+ affinity column chromatography and Mono Q column chromatography. A 5 µg aliquot of each purified recombinant protein was electrophoresed on a 12.5% polyacrylamide gel and proteins were visualized by staining with Coomassie brilliant blue R-250. Lane 1, molecular weight marker; lane 2, rRGA1; lane 3, rG48V; lane 4, rR191C; lane 5, rQ223L; lane 6, rA356S. (B) Time course analysis of GTPase hydrolysis by purified recombinant proteins. All recombinant proteins were incubated with [γ-32P]GTP at 25°C and the reaction was terminated at the indicated times. Released 32P was quantified with a liquid scintillation spectrometer. Data shown represent the means of duplicate measurements. rRGA1 (filled diamond), rG48V (open square), rR191C (filled square), rQ223L (filled triangle), rA356S (open circle). (C) [γ-32P]GTP-binding of purified recombinant proteins. The purified recombinant proteins were incubated with [γ-32P]GTP for 10 min, and then each of the incubation mixtures was applied to a Sephadex G 50 column to remove unbound [γ-32P]GTP from protein-bound [γ-32P]GTP. The protein fractions obtained in this way were incubated further with the reaction buffer and the reactions were terminated at the indicated times. rRGA1 (filled diamond), rG48V (open square), rR191C (filled square), rQ223L (closed triangle), rA356S (open circle). Open in new tabDownload slide Fig. 2 Purification of recombinant α subunits from the total lysates of E. coli, and GTPase and GTP-binding activities of recombinant proteins. (A) SDS–PAGE. rRGA1, rG48V, rR191C and rA356S were purified by Ni2+ affinity column chromatography and Mono Q column chromatography. A 5 µg aliquot of each purified recombinant protein was electrophoresed on a 12.5% polyacrylamide gel and proteins were visualized by staining with Coomassie brilliant blue R-250. Lane 1, molecular weight marker; lane 2, rRGA1; lane 3, rG48V; lane 4, rR191C; lane 5, rQ223L; lane 6, rA356S. (B) Time course analysis of GTPase hydrolysis by purified recombinant proteins. All recombinant proteins were incubated with [γ-32P]GTP at 25°C and the reaction was terminated at the indicated times. Released 32P was quantified with a liquid scintillation spectrometer. Data shown represent the means of duplicate measurements. rRGA1 (filled diamond), rG48V (open square), rR191C (filled square), rQ223L (filled triangle), rA356S (open circle). (C) [γ-32P]GTP-binding of purified recombinant proteins. The purified recombinant proteins were incubated with [γ-32P]GTP for 10 min, and then each of the incubation mixtures was applied to a Sephadex G 50 column to remove unbound [γ-32P]GTP from protein-bound [γ-32P]GTP. The protein fractions obtained in this way were incubated further with the reaction buffer and the reactions were terminated at the indicated times. rRGA1 (filled diamond), rG48V (open square), rR191C (filled square), rQ223L (closed triangle), rA356S (open circle). Open in new tabDownload slide Fig. 3 Transgenic rice plants expressing Q223L. (A) Schematic diagrams of the structures of the chimeric gene, ProRGA1:QL. The C-terminal part of mutagenized Q223L cDNA, which included the site of animo acid exchange, was fused to the RGA1 gene at the third exon. (B) Photograph of rice plants after flowering. A chimeric gene, ProRGA1:QL, was introduced into d1, a rice mutant defective in the α-subunit gene, by the Agrobacterium-mediated transformation method. d1, a rice mutant defective in the α-subunit gene; NC, normal cultivar; QL/d1, transformants with a chimeric gene, ProRGA1:QL, introduced into d1. (C) Western blot analysis. Plasma membrane proteins from d1, NC and QL/d1 were separated by SDS–PAGE and identified on Western blots using anti-α antibody. The position of the RGA1 and Q223L proteins is indicated by an arrow. (D) Photograph of rice seed of NC, d1 and QL/d1. Unhulled and hulled seeds are shown in the upper and lower parts, respectively. Open in new tabDownload slide Fig. 3 Transgenic rice plants expressing Q223L. (A) Schematic diagrams of the structures of the chimeric gene, ProRGA1:QL. The C-terminal part of mutagenized Q223L cDNA, which included the site of animo acid exchange, was fused to the RGA1 gene at the third exon. (B) Photograph of rice plants after flowering. A chimeric gene, ProRGA1:QL, was introduced into d1, a rice mutant defective in the α-subunit gene, by the Agrobacterium-mediated transformation method. d1, a rice mutant defective in the α-subunit gene; NC, normal cultivar; QL/d1, transformants with a chimeric gene, ProRGA1:QL, introduced into d1. (C) Western blot analysis. Plasma membrane proteins from d1, NC and QL/d1 were separated by SDS–PAGE and identified on Western blots using anti-α antibody. The position of the RGA1 and Q223L proteins is indicated by an arrow. (D) Photograph of rice seed of NC, d1 and QL/d1. Unhulled and hulled seeds are shown in the upper and lower parts, respectively. Table 1 Length and width of unhulled rice seeds Normal cultivar d1 QL/d1 10 27 33 43 46 Length 7.59 ± 0.44 4.79 ± 0.43 7.78 ± 0.36 7.98 ± 0.47 7.88 ± 0.41 7.77 ± 0.34 8.01 ± 0.36 Width 3.68 ± 0.35 3.54 ± 0.36 3.65 ± 0.34 3.72 ± 0.37 3.86 ± 0.33 3.51 ± 0.32 3.79 ± 0.36 Normal cultivar d1 QL/d1 10 27 33 43 46 Length 7.59 ± 0.44 4.79 ± 0.43 7.78 ± 0.36 7.98 ± 0.47 7.88 ± 0.41 7.77 ± 0.34 8.01 ± 0.36 Width 3.68 ± 0.35 3.54 ± 0.36 3.65 ± 0.34 3.72 ± 0.37 3.86 ± 0.33 3.51 ± 0.32 3.79 ± 0.36 Length and width are shown in mm. Data are the averages of 10 plants (±SD). Open in new tab Table 1 Length and width of unhulled rice seeds Normal cultivar d1 QL/d1 10 27 33 43 46 Length 7.59 ± 0.44 4.79 ± 0.43 7.78 ± 0.36 7.98 ± 0.47 7.88 ± 0.41 7.77 ± 0.34 8.01 ± 0.36 Width 3.68 ± 0.35 3.54 ± 0.36 3.65 ± 0.34 3.72 ± 0.37 3.86 ± 0.33 3.51 ± 0.32 3.79 ± 0.36 Normal cultivar d1 QL/d1 10 27 33 43 46 Length 7.59 ± 0.44 4.79 ± 0.43 7.78 ± 0.36 7.98 ± 0.47 7.88 ± 0.41 7.77 ± 0.34 8.01 ± 0.36 Width 3.68 ± 0.35 3.54 ± 0.36 3.65 ± 0.34 3.72 ± 0.37 3.86 ± 0.33 3.51 ± 0.32 3.79 ± 0.36 Length and width are shown in mm. Data are the averages of 10 plants (±SD). Open in new tab Table 2 Weight of rice seeds Normal cultivar d1 QL/d1 10 27 33 43 46 Unhulled seed 26.0 ± 1.03 14.8 ± 0.87 29.4 ± 1.26 30.4 ± 1.16 30.1 ± 1.09 29.3 ± 1.54 30.6 ± 1.47 Hull 4.50 ± 0.41 3.50 ± 0.49 4.70 ± 0.52 5.41 ± 0.43 5.22 ± 0.61 5.01 ± 0.52 5.92 ± 0.41 Hulled seed 21.5 ± 1.04 11.3 ± 0.81 24.7 ± 1.23 25.0 ± 1.18 24.9 ± 1.11 24.3 ± 1.42 24.7 ± 1.39 Normal cultivar d1 QL/d1 10 27 33 43 46 Unhulled seed 26.0 ± 1.03 14.8 ± 0.87 29.4 ± 1.26 30.4 ± 1.16 30.1 ± 1.09 29.3 ± 1.54 30.6 ± 1.47 Hull 4.50 ± 0.41 3.50 ± 0.49 4.70 ± 0.52 5.41 ± 0.43 5.22 ± 0.61 5.01 ± 0.52 5.92 ± 0.41 Hulled seed 21.5 ± 1.04 11.3 ± 0.81 24.7 ± 1.23 25.0 ± 1.18 24.9 ± 1.11 24.3 ± 1.42 24.7 ± 1.39 Weight is shown in mg. Data are the averages of 10 plants (±SD). Open in new tab Table 2 Weight of rice seeds Normal cultivar d1 QL/d1 10 27 33 43 46 Unhulled seed 26.0 ± 1.03 14.8 ± 0.87 29.4 ± 1.26 30.4 ± 1.16 30.1 ± 1.09 29.3 ± 1.54 30.6 ± 1.47 Hull 4.50 ± 0.41 3.50 ± 0.49 4.70 ± 0.52 5.41 ± 0.43 5.22 ± 0.61 5.01 ± 0.52 5.92 ± 0.41 Hulled seed 21.5 ± 1.04 11.3 ± 0.81 24.7 ± 1.23 25.0 ± 1.18 24.9 ± 1.11 24.3 ± 1.42 24.7 ± 1.39 Normal cultivar d1 QL/d1 10 27 33 43 46 Unhulled seed 26.0 ± 1.03 14.8 ± 0.87 29.4 ± 1.26 30.4 ± 1.16 30.1 ± 1.09 29.3 ± 1.54 30.6 ± 1.47 Hull 4.50 ± 0.41 3.50 ± 0.49 4.70 ± 0.52 5.41 ± 0.43 5.22 ± 0.61 5.01 ± 0.52 5.92 ± 0.41 Hulled seed 21.5 ± 1.04 11.3 ± 0.81 24.7 ± 1.23 25.0 ± 1.18 24.9 ± 1.11 24.3 ± 1.42 24.7 ± 1.39 Weight is shown in mg. Data are the averages of 10 plants (±SD). Open in new tab Table 3 Length of ear and internodes of rice plants after flowering Normal cultivar d1 QL/d1 10 27 33 43 46 Ear 19.7 ± 1.8 13.1 ± 1.1 18.8 ± 1.8 20.6 ± 1.9 20.4 ± 1.6 18.6 ± 1.2 20.9 ± 1.6 First internode 32.6 ± 3.7 17.9 ± 1.4 34.8 ± 1.8 35.8 ± 3.6 36.4 ± 1.9 33.7 ± 2.1 35.2 ± 2.5 Second internode 16.3 ± 1.9 6.5 ± 0.5 16.7 ± 1.9 17.5 ± 1.5 17.3 ± 1.0 16.8 ± 1.1 18.2 ± 1.4 Third internode 8.9 ± 1.8 3.0 ± 1.0 8.3 ± 1.4 7.6 ± 1.6 7.7 ± 0.9 8.5 ± 1.6 8.8 ± 2.3 Fourth internode 4.0 ± 1.9 0.8 ± 0.5 3.0 ± 1.4 3.2 ± 1.7 3.5 ± 1.8 4.6 ± 2.0 3.6 ± 2.5 Total 82.0 ± 6.8 41.3 ± 2.2 82.2 ± 5.1 85.3 ± 7.6 86.0 ± 4.9 83.0 ± 4.2 87.3 ± 4.6 Normal cultivar d1 QL/d1 10 27 33 43 46 Ear 19.7 ± 1.8 13.1 ± 1.1 18.8 ± 1.8 20.6 ± 1.9 20.4 ± 1.6 18.6 ± 1.2 20.9 ± 1.6 First internode 32.6 ± 3.7 17.9 ± 1.4 34.8 ± 1.8 35.8 ± 3.6 36.4 ± 1.9 33.7 ± 2.1 35.2 ± 2.5 Second internode 16.3 ± 1.9 6.5 ± 0.5 16.7 ± 1.9 17.5 ± 1.5 17.3 ± 1.0 16.8 ± 1.1 18.2 ± 1.4 Third internode 8.9 ± 1.8 3.0 ± 1.0 8.3 ± 1.4 7.6 ± 1.6 7.7 ± 0.9 8.5 ± 1.6 8.8 ± 2.3 Fourth internode 4.0 ± 1.9 0.8 ± 0.5 3.0 ± 1.4 3.2 ± 1.7 3.5 ± 1.8 4.6 ± 2.0 3.6 ± 2.5 Total 82.0 ± 6.8 41.3 ± 2.2 82.2 ± 5.1 85.3 ± 7.6 86.0 ± 4.9 83.0 ± 4.2 87.3 ± 4.6 The length is shown in cm. Data are the averages of 10 plants (±SD). Open in new tab Table 3 Length of ear and internodes of rice plants after flowering Normal cultivar d1 QL/d1 10 27 33 43 46 Ear 19.7 ± 1.8 13.1 ± 1.1 18.8 ± 1.8 20.6 ± 1.9 20.4 ± 1.6 18.6 ± 1.2 20.9 ± 1.6 First internode 32.6 ± 3.7 17.9 ± 1.4 34.8 ± 1.8 35.8 ± 3.6 36.4 ± 1.9 33.7 ± 2.1 35.2 ± 2.5 Second internode 16.3 ± 1.9 6.5 ± 0.5 16.7 ± 1.9 17.5 ± 1.5 17.3 ± 1.0 16.8 ± 1.1 18.2 ± 1.4 Third internode 8.9 ± 1.8 3.0 ± 1.0 8.3 ± 1.4 7.6 ± 1.6 7.7 ± 0.9 8.5 ± 1.6 8.8 ± 2.3 Fourth internode 4.0 ± 1.9 0.8 ± 0.5 3.0 ± 1.4 3.2 ± 1.7 3.5 ± 1.8 4.6 ± 2.0 3.6 ± 2.5 Total 82.0 ± 6.8 41.3 ± 2.2 82.2 ± 5.1 85.3 ± 7.6 86.0 ± 4.9 83.0 ± 4.2 87.3 ± 4.6 Normal cultivar d1 QL/d1 10 27 33 43 46 Ear 19.7 ± 1.8 13.1 ± 1.1 18.8 ± 1.8 20.6 ± 1.9 20.4 ± 1.6 18.6 ± 1.2 20.9 ± 1.6 First internode 32.6 ± 3.7 17.9 ± 1.4 34.8 ± 1.8 35.8 ± 3.6 36.4 ± 1.9 33.7 ± 2.1 35.2 ± 2.5 Second internode 16.3 ± 1.9 6.5 ± 0.5 16.7 ± 1.9 17.5 ± 1.5 17.3 ± 1.0 16.8 ± 1.1 18.2 ± 1.4 Third internode 8.9 ± 1.8 3.0 ± 1.0 8.3 ± 1.4 7.6 ± 1.6 7.7 ± 0.9 8.5 ± 1.6 8.8 ± 2.3 Fourth internode 4.0 ± 1.9 0.8 ± 0.5 3.0 ± 1.4 3.2 ± 1.7 3.5 ± 1.8 4.6 ± 2.0 3.6 ± 2.5 Total 82.0 ± 6.8 41.3 ± 2.2 82.2 ± 5.1 85.3 ± 7.6 86.0 ± 4.9 83.0 ± 4.2 87.3 ± 4.6 The length is shown in cm. 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Plant and Cell PhysiologyOxford University Press

Published: Feb 1, 2005

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