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American Society of Plant Biologists
Overexpression of the Heterotrimeric G-Protein
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODSNOTE ADDED IN PROOFReferences |
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subunit of the heterotrimeric G-protein under the control of a glucocorticoid-inducible promoter. With the conditional overexpression of either the wild type or a constitutively active version of Arabidopsis G, transgenic seedlings exhibited a hypersensitive response to light. This enhanced light sensitivity was more exaggerated in a relatively lower intensity of light and was observed in white light as well as far-red, red, and blue light conditions. The enhanced responses in far-red and red light required functional phytochrome A and phytochrome B, respectively. Furthermore, the response to far-red light depended on functional FHY1 but not on FIN219 and FHY3. This dependence on FHY1 indicates that the Arabidopsis G protein may act only on a discrete branch of the phytochrome A signaling pathway. Thus, our results support the involvement of a heterotrimeric G-protein in the light regulation of Arabidopsis seedling development. |
INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSNOTE ADDED IN PROOFReferences |
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), and a maternal G-protein was found to be important for the orientation of cell division axes in early Caenorhabditis elegans embryos (Zwaal et al., 1996). In most cases, heterotrimeric G-proteins are involved in the propagation of perceived signals.
Perception and interpretation of environmental cues are the most important processes for immotile organisms such as plants. Heterotrimeric G-proteins have been implicated in several processes during growth and development, and they transduce extracellular environmental signals to the cell. In general, heterotrimeric G-proteins consist of three subunits: , 
, and 
. Analysis of the complete genome sequence (Arabidopsis Genome Initiative, 2000) indicates that the Arabidopsis genome contains only a single G gene, previously designated AtGPA1 (Ma et al., 1990), and a single G gene, designated AGB1 (Weiss et al., 1994). Genes homologous with Arabidopsis G and G also have been found in other plant species. The Arabidopsis genome sequencing program did not identify obvious G sequences in Arabidopsis, although there is some biochemical evidence to suggest that plants may have a membrane-associated G-like protein (Obrdlik et al., 2000). Recently, the Arabidopsis G subunit was identified by a yeast two-hybrid screen using G as bait (Mason and Botella, 2001). It was shown that G also is encoded by a single copy gene in Arabidopsis.
In plants, the G subunit is the most intensively studied of the three subunits of the complex. AtGPA1 of Arabidopsis encodes a protein of 383 amino acids and 45 kD. This protein shows 36% identity and 73% similarity to Gi of mammals and Gt of vertebrates (Ma, 1994). It also has the conserved arginine residue that is predicted to be the target site for cholera toxin in vertebrate G (Ma et al., 1990). The transcript is most abundant in vegetative tissue (Huang et al., 1994). The G protein is detected during all stages of Arabidopsis development except in mature seed and is found in virtually all parts, from roots and leaves to the reproductive organs (Weiss et al., 1993). It is closely associated with the endoplasmic reticulum and the plasma membrane (Weiss et al., 1997). In rice, G antisense expressor lines show a dwarf phenotype in overall morphology at all stages of development (Fujisawa et al., 1999). In addition, five alleles of dwarf1 (d1), which was classified originally as a gibberellin-insensitive mutant, have been found to have mutations in a heterotrimeric G gene (Ashikari et al., 1999; Fujisawa et al., 1999; Ueguchi-Tanaka et al., 2000), suggesting an involvement in gibberellin signal transduction. Various drug application studies have suggested that G is involved in gibberellin induction of the -amylase gene in oat aleurone cells (Jones et al., 1998). In addition, the regulation of stomata opening (Assmann, 1996), pollen tube elongation in lily (Ma et al., 1999), and light signaling pathways in tomato cells (Neuhaus et al., 1993) have been reported to be regulated by heterotrimeric G-proteins. On the basis of cell biological and pharmacological studies, it has been proposed that a heterotrimeric G-protein acts downstream of phytochrome A and phytochrome B photoreceptors (Neuhaus et al., 1993; Kunkel et al., 1996).
Among all of the known photoreceptors in plants, the red (R) and far-red (FR) light–absorbing phytochromes are the most well-characterized molecules. They are encoded by a family of five genes (PHYA to PHYE) in Arabidopsis (Sharrock and Quail, 1989). The proteins encoded by these genes covalently attach a tetrapyrrole chromophore to form functional phytochromes. Phytochrome is synthesized in darkness as an R light–absorbing form and is converted to the FR light–absorbing form (Pfr) upon R light absorption. Although phytochromes have partially overlapping functions, it was established that phytochrome A (phyA) is the primary photoreceptor for FR light–mediated inhibition of hypocotyl elongation (Nagatani et al., 1993; Parks and Quail, 1993; Whitelam et al., 1993), whereas phytochrome B (phyB) was found to be the primary photoreceptor for R light–mediated inhibition of hypocotyl elongation (Reed et al., 1993). phyA also mediates, in addition to hypocotyl elongation, other FR light–regulated processes, such as the induction of germination, the induction of light-regulated genes, and an FR light block of the greening response.
The FR light inhibition of hypocotyl elongation has been used as a marker for mutations defective in phyA-specific signaling components. The first two mutants identified as such are fhy1-1 and fhy3-1 (Whitelam et al., 1993). Although both mutants are defective in phytochrome-mediated FR light inhibition of hypocotyl elongation, they still possess some phyA-mediated responses in FR light. Studies of these mutants have suggested that the phyA signal transduction is branched. Recently, a number of additional mutants that are defective in FR light regulation of hypocotyl elongation have been isolated. These are fin2, spa1, far1, pat1, fin219, and hfr1/rsf1/rep1 (Soh et al., 1998, 2000; Hoecker et al., 1999; Hudson et al., 1999; Bolle et al., 2000; Fairchild et al., 2000; Fankhauser and Chory, 2000; Hsieh et al., 2000). The responsible genes have been identified except for fin2. These genes encode both nuclear (SPA1, FAR1, and HFR1) and cytoplasmic (PAT1 and FIN219) proteins. In addition, yeast two-hybrid screens also have identified proteins that interact directly with phyA and/or phyB. These are PIF3, PKS1, and NDPK2 (Ni et al., 1998; Choi et al., 1999; Fankhauser et al., 1999). Interestingly, PIF3 has a bHLH domain that binds to DNA and was shown to act as a transcription factor (Martinez-Garcia et al., 2000). The HFR1 protein also possesses a bHLH domain and was shown to interact with the PIF3 protein and possibly form a heterodimer (Fairchild et al., 2000). It was demonstrated that both phyA and phyB molecules are imported from the cytoplasm into the nucleus upon R and/or FR irradiation (Kircher et al., 1999; Yamaguchi et al., 1999). Thus, it is plausible that phytochromes could serve as a direct regulator of these transcription factors. However, phytochrome signaling also must involve cytoplasmic events, because PKS1, PAT1, and FIN219 proteins were found primarily in the cytoplasm. In addition, it has been suggested that the secondary messengers, such as cytosolic free calcium and cyclic GMP, could be involved in the phytochrome signaling pathway and act downstream of the heterotrimeric G-protein (Neuhaus et al., 1993; Bowler et al., 1994).
In this study, we designed a transgenic approach to examine whether a heterotrimeric G-protein is involved in the phytochrome signal transduction pathway. We have generated transgenic Arabidopsis plants that are inducible overexpressors of the heterotrimeric G protein, AtGPA1. The transgenes were placed under a glucocorticoid-inducible promoter (Aoyama and Chua, 1997). Our analyses of these transgenic plants suggest that G overexpression can result in enhanced responses to light and that these phenotypes are dependent on functional phyA and phyB. Thus, this study provides physiological evidence of the involvement of heterotrimeric G-protein in light-regulated seedling development.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSNOTE ADDED IN PROOFReferences |
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-Inducible Arabidopsis Transgenic Lines; Ma et al., 1990) and a constitutively active form of AtGPA1 (cG; see Methods), were placed under the control of a glucocorticoid-inducible promoter (Aoyama and Chua, 1997) that could be activated by exogenously applied dexamethasone (DEX). A potential constitutively active form of AtGPA1 was generated by a point mutation of Glu-222 to Leu. This mutation was shown to disable the GTPase activity of G so that, once activated, it is locked as an active molecule (Aharon et al., 1998). Both transgenes were introduced into wild-type Arabidopsis.
The transgenic T2 lines that had segregated to give 75% hygromycin-resistant plants and thus were likely to contain a single insertion of the transgene were selected (15 for wG and 17 for cG). These lines were subjected to a screen on a plate containing DEX. These plates were placed under dim white light (10 µmol·m-2·sec-1) for 5 days, and the seedlings were compared with wild-type or empty vector transgenic plants grown on the same plates. Under these conditions, 5 of 15 lines of wG and 8 of 17 lines of cG segregated to give a population with 75% of the seedlings having a short hypocotyl phenotype relative to the wild type and the empty vector controls. The hygromycin resistance cosegregated tightly with the shorter hypocotyl phenotype. We chose one of each of the wG (line 1.1) and cG (line 2.1) lines for further analysis, and these two lines will be referred to as wG and cG, respectively.
To avoid the artificial influence of DEX described previously (Kang et al., 1999), we applied various concentrations of DEX to the transgenic plants and examined their phenotypes under these conditions. False traits, such as a stunted root and half-closed cotyledons at the seedling stage, were observed regularly at DEX concentrations greater than 1 µM and also were observed in the empty vector control plants. To avoid the false phenotype, we routinely used DEX concentrations between 10 and 100 nM. Within this range of DEX concentrations, we observed no false phenotypes in our selected transgenic lines or in wild-type seedlings (data not shown). The optimum concentration of DEX for each line was decided on the basis of the level of G protein expression.
wG and cG Lines Overexpress G Protein by DEX Induction
Both wG and cG lines grown in the presence of DEX (30 nM for wG and 70 nM for cG; see Methods) overexpressed the G protein. The wG and cG seedlings grown on plates with these concentrations of DEX had increased levels of G protein relative to the empty vector control transgenic line (VE) and wild-type seedlings both in darkness and under continuous white light (Figures 1A and 1B). Under these conditions, the transgenic lines expressed at least two times more G protein in both wG and cG seedlings than did the wild type and the control VE line.
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and cG dark-grown seedlings. However, in the VE control or wild-type seedlings, the monomeric form was barely detectable (Figure 1C; data not shown). In light-grown seedlings, there was a higher ratio of monomeric versus complex form in all lines, and the monomeric form was detectable in the VE control line in the light (Figure 1D). Overall, the overexpressor lines in general had a higher monomer-to-complex ratio than the VE controls. Because the GTP binding form of G is thought to be a monomer, it is possible that the monomeric protein we detected in the light-grown control plants was the active G.
G Overexpressors Show Exaggerated Light Responses
When the wG and cG lines were grown under continuous irradiation with white light (10 µmol·m-2·sec-1) for 5 days in the presence of DEX, the hypocotyl length of these lines was reduced to 
60% of that of the control plants (Figures 2A and 2D). However, dark-grown seedlings exhibited no significant difference between the VE control and the G overexpression lines (Figure 2D). Given that the dark-grown seedlings expressed as much G protein as the light-grown seedlings after DEX treatment (Figure 1A), these results indicate that the observed hypocotyl phenotype is light dependent.
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and cG lines were sown on plates containing 30 and 70 nM DEX, respectively (these concentrations give a similar amount of G overexpression) (Figures 1A and 1B). When these plants were exposed to very low fluence light, both wG and cG seedlings started to show increased reduction in hypocotyl length compared with the VE control and the wild type. The fluence response curves of both wG and cG lines were steeper than those of the control plants in the low light range. The differences between the wild-type, control VE, and G transgenic plants gradually became smaller when a higher intensity of light was applied, and the differences were no longer detectable at a light fluence of 160 µmol·m-2·sec-1 (Figures 3A and 3B).
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lines in terms of hypocotyl length, cotyledons of wG and cG lines were much smaller than those of the control (Figure 2B). Interestingly, the chlorophyll content per seedling of the G lines also was reduced to 50% of the level of the control plants under this high light fluence (Figure 2E), but the chlorophyll a/b ratios of these seedlings remained the same as in the VE control (data not shown). This reduction of chlorophyll content correlated with small cotyledons, which may contain fewer cells or smaller cells with fewer chloroplasts. The transcript levels of light-induced genes such as CAB and RBCS were slightly lower in the G-overexpressing plant than in the VE control (Figure 2C).
The Short Hypocotyl Phenotype of G Overexpression Lines Is a Result of Reduced Hypocotyl Cell Elongation
The smaller cotyledons and shorter hypocotyls in the G lines relative to the control VE line could be a result of a reduced number of cells on the hypocotyl or an effect on cell elongation. Because Arabidopsis seedling development is completed in embryogenesis during seed maturation, the number of cells per seedling would be predetermined. It has been reported that epidermal or cortical cell divisions are insignificant for Arabidopsis hypocotyl elongation in dark- and light-grown seedlings (Gendreau et al., 1997). To determine whether the G overexpressor lines had affected hypocotyl cell elongation or cell division, we counted the number of hypocotyl epidermal cells of both G lines and the control seedlings grown for 5 days under white light (10 µmol· m-2·sec-1). The light-grown Arabidopsis hypocotyl epidermis consists of two distinct files of mostly alternating cells (Gendreau et al., 1997). One type of file contains larger and more protruding cells, whereas the other, which is made up of burrowed cells, lies between the protruding cell files (see scheme of the transverse section of hypocotyl cells in Figure 4A). Stomata structures are found only in burrowed cell files, and there is no noticeable cell differentiation in the protruding cell files.
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, 21.7 ± 2.0 (n = 15) cells in cG, and 22.3 ± 1.7 (n = 12) cells in control VE seedlings in the presence of DEX. The cell numbers of the protruding cell files between these lines were essentially the same, although there was a nearly twofold difference in the hypocotyl length (Figures 2A and 2C). These cell numbers are similar to those reported previously for Arabidopsis seedlings (Gendreau et al., 1997). Therefore, we conclude that there is no significant difference between the G lines and the control seedlings in terms of the number of protruding epidermal cells in the hypocotyl and that the reduction of hypocotyl length observed was caused by a reduction of cell elongation in the G seedlings.
G Lines Have an Increased Number of Stomata in the Hypocotyl Epidermis
We also observed a greater degree of stomatal differentiation on burrowed cell files of the hypocotyl epidermis in G-induced lines compared with those of the control plants (Figures 4B, 4C, and 4D). The average number of stomata structures per burrowed cell file was 5.0 ± 1.1 (n = 10) in the wG line, 5.5 ± 0.7 (n = 10) in the cG line, and 1.1 ± 0.3 (n = 10) in the control line. One factor known to stimulate stomatal differentiation is ethylene (Serna and Fenoll, 1997). It is possible that the increased level of G influenced the phytohormone balance in these plants. Another factor reported to affect stomatal differentiation is light. The frequency of stomata is usually higher in plants grown in high photon flux density than in plants grown in shade (Willmer and Fricker, 1996). Thus, this high frequency of stomata differentiation in G-induced lines could be a result of an exaggerated light response caused by the overexpression of G.
G Overexpression Does Not Affect the Gibberellic Acid Stimulation of Hypocotyl Elongation
It has been reported that the G knockout and the G antisense expressors of rice have a dwarf phenotype and are insensitive to the exogenous application of gibberellin (Ashikari et al., 1999; Fujisawa et al., 1999; Ueguchi-Tanaka et al., 2000). Although generating the loss-of-function rice G mutants and our G overexpression mutant results in opposite effects at the genetic level, the phenotype of these two plants is similar. It is known that gibberellin is produced more actively in the apical meristem of light-grown plants than in that of dark-grown plants. It is possible that the light-dependent short-hypocotyl phenotype could be a consequence of insensitivity to this hormone produced in the light.
To determine whether the overexpression of G in Arabidopsis causes insensitivity to gibberellin, we applied gibberellic acid (GA3) to the cG overexpressor in the presence of DEX (Figure 5B). An increase in hypocotyl length was detected in both controls and the cG line, and the extent of this increase was indistinguishable in all lines. In the absence of DEX, the phenotype of the cG transgenic seedlings also was indistinguishable from that of the wild type and the VE control (Figure 5A). The response of the wG line to GA3 was similar to that of the cG line (data not shown). This result indicates that the G overexpressors retain the full capacity to respond to GA3.
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mutants and our G overexpression (gain-of-function) mutants are opposite in nature, both of them exhibited dwarf phenotypes, albeit at distinct stages. It is not clear why there is this difference between rice and Arabidopsis in their G-protein involvement in gibberellin response. It is possible that either the distinct species or the developmental stages examined may be responsible for the observed inconsistency.
The G Overexpressors Affect the Blue (B), R, and FR Light–Mediated Inhibition of Hypocotyl Elongation
We then analyzed the wG and cG lines under narrow wavelengths of blue (B), R, or FR light to examine whether the light-dependent G phenotype is dependent on a specific photoreceptor. The plants were grown under continuous irradiation with each light source for 5 days on DEX plates. Under the light conditions we used (B, 1.8 µmol·m-2· sec-1; R, 8.9 µmol·m-2·sec-1; FR, 13.2 µmol·m-2·sec-1), both lines showed a shorter hypocotyl phenotype relative to the control plants under all three light conditions (Figure 5C). These results suggest that G modulates signals from both B light receptors and R/FR light receptor phytochromes. Because microinjection studies using tomato seedlings indicated that the heterotrimeric G-protein may act downstream of phyA and phyB photoreceptors (Neuhaus et al., 1993; Kunkel et al., 1996), we analyzed the effect of G overexpression on phyA, phyB, and CRY1 signal transduction.
G Overexpressors Required Functional phyA for Their Enhancement of the FR Light–Mediated Inhibition of Hypocotyl Elongation
To examine the role of G in phyA signal transduction, we first examined its dependence on the phyA receptor. Both types of G-inducible lines were crossed to the phyA null mutant independently, and plants homozygous for the phyA mutation carrying the transgenes were obtained. If an input from the photoreceptor is required to generate the G overexpression phenotype, phyA must be required to obtain the exaggerated hypocotyl reduction phenotype in FR light. If this hypothesis is correct, G overexpression in the phyA mutant background would not show a hypocotyl reduction under FR light. Indeed, neither of the G lines in the phyA null mutant background exhibited the typical G-induced exaggeration of hypocotyl inhibition under FR light (Figures 6A and 6B).
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Could Be Involved in a Branch of the phyA-Mediated FR Light Signal Transduction Pathway to the downstream events of phyA signal transduction, we also crossed the G-inducible lines to three representative downstream signaling mutants (fhy1-1, fhy3-1, and fin219). It has been shown that the phyA signal transduction mutants fhy3-1 and fhy1-1 define a branch point in the phyA signaling pathway and are defective in subsets of FR light responses (Barnes et al., 1996; Yanovsky et al., 2000). Interestingly, we detected no G overexpression effect in the fhy1-1 background under FR light. However, we observed a normal G overexpression effect in both the fhy3-1 and fin219 backgrounds. Our results suggest that the G overexpression phenotype requires a functional phyA receptor as well as FHY1, but that it does not require FHY3 or FIN219. This finding indicates that G may be involved in only a branch of the phyA signaling pathway.
The G-overexpressing seedlings in all mutant backgrounds, including phyA and fhy1-1, showed the exaggerated reduction of hypocotyl response under R light (Figures 6C and 6D). This finding indicates that the G overexpression phenotype under R light requires neither phyA nor FHY1. In other words, under R light, G most likely interacts with a pathway that does not require phyA or FHY1.
G Overexpressors Required Functional phyB for Their Enhancement of the R Light–Mediated Inhibition of Hypocotyl Elongation
To determine whether phyB is required for the observed enhancement of R light inhibition of hypocotyl elongation caused by the G overexpressors, we crossed the transgenes into a phyB mutant background. As shown in Figure 7A, the G overexpression phenotype under R light was not present in the phyB mutant background. Therefore, the phyB receptor is needed for the effect of the G overexpressors on the R light–mediated inhibition of hypocotyl elongation. It is important to note that both wG and cG in the phyB background still manifest the G overexpression effect under FR light (Figure 7B), indicating that phyB is not involved in the corresponding FR effect of the G overexpressors.
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Overexpression Does Not Affect phyB-Mediated End-of-Day FR Response lines retain the EODFR light–dependent stimulation of hypocotyl elongation. The rate of EODFR light–stimulated hypocotyl elongation in G lines was equivalent to that in the control plants, indicating that these seedlings retain the capacity to increase their hypocotyl length after EODFR treatment. The effect of the EODFR treatment could be reversed completely with an R light pulse applied immediately after the EODFR light treatment, regardless of the presence of G overexpression. This result also suggests that G is not involved in this phyB-mediated developmental response.
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Overexpression Does Not Affect CRY1-Mediated B Light Inhibition of Hypocotyl Elongation transgene was crossed into cry1, a B light receptor mutant background, and its involvement in CRY1-mediated light signal transduction was examined. cry1 seedlings overexpressing G had short hypocotyls in B and FR light (Figure 9). This result indicates that the G signaling pathway is not involved in the B light receptor CRY1 pathway. It is possible that G might be used to modulate other photoreceptors responsible for the B light effect.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSNOTE ADDED IN PROOFReferences |
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protein in modulating phytochrome-mediated light regulation of hypocotyl elongation during seedling development. Significantly, the enhancement of FR and R light inhibition of hypocotyl elongation by conditional G overexpression depends entirely on functional phyA and phyB photoreceptors, respectively.
G May Be Involved in Branches of the phyA and phyB Signaling Pathways
We crossed the G-inducible lines (both wG and cG) to phytochrome signaling mutants (fhy1-1, fin219, and fhy3-1) as well as to a phyA null mutant and examined the effect of the G overexpression on phyA signal transduction. The phenotype of the G-overexpressing phyA seedlings was identical to that of the phyA mutant (Figures 6A and 6B), indicating that the phyA mutation prevented G from overactivating the FR light–dependent signaling pathway. It is known that the fhy1-1 mutant is blocked in only a subset of phyA-mediated responses at the physiological level (Johnson et al., 1994). It also is deficient in the regulation of only a subset of genes such as CHS, with CAB gene expression remaining relatively unaffected in FR light (Barnes et al., 1996). Therefore, it was proposed that fhy1-1 may define a component of the cyclic GMP–dependent pathway that regulates CHS (Barnes et al., 1997).
Recent physiological studies also have supported the idea that FHY1 and FHY3 may act in discrete pathways downstream of phyA (Yanovsky et al., 2000). Interestingly, as with the phyA mutant background, overexpression of G no longer conferred the increased hypocotyl reduction phenotype in the fhy1-1 mutant background (Figure 6). This finding suggests that fhy1-1 has a defect in the same signal transduction pathway in which G is involved. In contrast to the phyA and fhy1-1 mutants, two other phyA signal transduction mutations (fin219 and fhy3-1) did not block the phenotype caused by G overexpression (Figure 6). It seems likely that the active G is involved in the pathway defined by FHY1 in mediating FR light responses (Figure 10A). The relationship between the G protein and FHY1 in this pathway—that is, the hierarchy of these molecules in this signaling process—is not clear. This will be an immediate question for future investigation.
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overexpression did not exhibit its phenotypic effect under R light in the phyB mutant background (Figure 7). This result indicates that phyB is essential for the G overexpression phenotype in R light. However, G overexpression had no effect on the EODFR response. This suggests that G most likely is not involved in this phyB-mediated response. Again, the heterotrimeric G-protein regulates only a subset of phyB-mediated responses (Figure 10B).
It is important to note that our gain-of-function studies do not allow an assessment of whether the G protein is part of the phyA signaling cascade. They only suggest that excess G protein could enhance the signal propagation for specific phyA- and phyB-mediated responses. Future studies with Arabidopsis G protein loss-of-function mutants will be required.
G Overexpression May Result in More G-Protein Activity in Arabidopsis Seedlings
The gel filtration data showed that there is a higher ratio of monomeric to complex G in light-grown VE control seedlings compared with seedlings grown in darkness (Figure 1D). Because the GTP binding form of G is thought to be a monomer, it is possible that the monomeric protein we detected in light-grown plants could be active G. In both wG- and cG-overexpressing seedlings, there were notable increases of the monomeric G protein under both dark and light growth conditions. Although more monomeric G in light-grown seedlings would be consistent with an enhanced light response, the increased monomeric G protein detected in the dark-grown G overexpressor seedlings did not result in any phenotypic consequences. Perhaps this excess amount of G cannot function in the regulation of hypocotyl elongation. It is possible that the monomeric forms we detected in G-overexpressing plants in darkness represent a pool of G that is not yet associated with the membrane and thus cannot be activated by an upstream signal. On the other hand, our data indicate that the effect of G on hypocotyl elongation requires functional photoreceptors and thus G activation. It is possible that the presence of active G in darkness would not be able to elicit any physiological effect without activated photoreceptors and their associated downstream signaling processes.
It is well documented that, in general, the activation of G-protein–coupled protein receptors initiates the dissociation of the heterotrimeric G-protein complex into a GTP-bound G subunit and a G complex and that each of these in turn binds and activates target enzymes, generating intracellular messengers. Significantly, the conformation of free G is identical to that of G in the heterotrimer, suggesting that G inhibits the interaction of G with its effectors or target molecules through the G binding site on G (Hamm, 1998). The crystal structure of the complex form of G and one of its effectors, adenylate cyclase, showed that the G activation site for the adenylate cyclase also is the site facing the G complex during the formation of the heterotrimeric G-protein complex (Bourne, 1997). Therefore, the active sites of both the G monomer and the G complex are suppressed by the formation of a trimer between these proteins. In this sense, both the G monomer and the G complex could be considered potentially active forms of the trimeric G-protein. Therefore, another possible explanation for the overexpressed G phenotype in the light would be the availability of the other subunits of the heterotrimer. It is possible that the dissociated G complex might be the active regulator for hypocotyl cell elongation.
Although the phenotypic difference between the wG and cG lines is not dramatic, cG seedlings were consistently shorter than wG seedlings (data not shown). Given that the overexpression levels of wG and cG were similar, as demonstrated by protein gel blot analysis, the overexpressed cG might be more active than wG. This effect could be attributable to the increased availability of the GTP-bound monomeric form of G caused by the constitutively active mutant of the cG protein.
A Possible Mechanism of Heterotrimeric G-Protein Activation through Phytochrome
In general, heterotrimeric G-proteins are activated by seven transmembrane G-protein–associated receptors. phyA and phyB are not membrane-bound or membrane-associated proteins. How could the phytochrome signal be coupled to the heterotrimeric G-protein?
By immunocytochemical and biochemical means, some phyA in the Pfr form has been found sequestered in membrane structures (reviewed in Kendrick and Kronenberg, 1994). It is possible that the Pfr form can be directed to the membrane upon activation and activate heterotrimeric G-proteins on the membrane. It was suggested that in Dictyostelium, G-protein activation is mediated by receptor-stimulated nucleoside diphosphate (NDP) kinase (Bominaar et al., 1993). Therefore, the NDP kinase 2 (NDPK2) that was found in a yeast two-hybrid assay to interact with phyA (Choi et al., 1999) could be a potential activator of the heterotrimeric G-protein when it is activated by photoreceptors. This is consistent with the fact that the active Pfr form of phyA interacts with NDPK2 to a significantly greater degree than does the R light–absorbing form. Considering the relatively insoluble nature of the Pfr form, NDPK2 might be recruited to the membrane by phytochrome upon activation. In support of this idea, R light–dependent phosphorylation of NDP kinase has been reported in pea (Tanaka et al., 1998). It is possible that phytochrome-dependent activation of NDP kinase that is sequestered by phytochrome to the membrane might activate G-proteins by increasing the level of GTP in their vicinity. This raises the possibility that G-proteins might interact directly with either phytochrome or NDPK2.
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METHODS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSNOTE ADDED IN PROOFReferences |
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cDNA was amplified by polymerase chain reaction using this cDNA library as a template with gene-specific primers (5'-CTCGAGATGGGCTTACTCTGCAGTAGAA-3' and 5'-ACTAGTTCATAAAAGGCCAGCCTCCAGTA-3') complementary to regions encoding the N and C termini of the protein. The constitutively active form of G was generated by changing Glu-222 to Leu by polymerase chain reaction–based mutagenesis using primers 5'-TGACGTGGGTGGACTGAGAAATGAGAGGAGG-3' and 5'-CCTCCTCTCATTTCTCAGTCC-ACCCACGTCA-3' in combination with the primer sets used to amplify G cDNA. These fragments were inserted into XhoI and SpeI sites in the pTA7002 vector (Aoyama and Chua, 1997) and transferred into Agrobacterium tumefaciens GV3011, which was used to transform Arabidopsis. The ecotype Wassilewskija was used for plant transformation; the same ecotype was transformed with the empty vector as the control. The transformants were selected for their hygromycin resistance, and the surviving seedlings were transferred to soil to obtain seed. In the next generation, the lines that segregated 25% hygromycin-sensitive seedlings (single locus lines) were selected for further analysis.
Finally, one line each from the wild type (wG) and the constitutively active type (cG) were selected for further biochemical and physiological analyses. For genetic crosses into photomorphogenic mutants, the phyA photoreceptor mutant phyA-101 as well as the phytochrome signal transduction mutants fhy1-1, fhy3-1, and fin219 were used. The phyA mutant is in the Nossen background, fhy1 is in Landsberg, and fhy3-1 and fin219 are in the Columbia ecotype. The phyB mutant is in the Landsberg background, and cry1-304 is in the Columbia background. Examination of multiple F2 progeny from these crosses indicated that the background ecotypes did not exhibit observable effects on the transgene phenotypes.
Plant Growth Conditions
Seed were surface sterilized and kept at 4°C in darkness for 1 day. The seed were sown on plates containing 0.8% agarose, 1% sucrose, 1 x Murashige and Skoog (1962) salts with vitamin mixture (Life Technologies, Rockville, MD), and 0.05% Mes; pH was adjusted with KOH to pH 5.8 unless stated otherwise. A water-soluble form of dexamethasone (DEX; Sigma) was added to the plates in the range of 10 to 100 nM. For the two lines selected for extensive analysis, optimal DEX concentrations (wG, 30 nM; cG, 70 nM) were determined and used throughout the studies. Control plants were sown on the same plates for both DEX concentrations. Because there was no significant difference between phenotypes of control plants grown on 30 and 70 nM DEX plates, all of the control data shown in the figures were taken from the 70 nM plates. The seed sown on the agar plates were kept at 4°C in darkness for 2 to 3 days and were exposed to continuous white light for 24 hr to initiate germination, unless stated otherwise.
Light Sources
Monochromatic blue, far-red (FR), and red (R) light were obtained by light-emitting diodes (model E-30; Percival Scientific, Boone, IA) at peak wavelengths of 470, 669, and 739 nm, respectively, with a half-bandwidth of 10 nm. The FR light source was covered with a plexiglass filter (FRF 700; Westlake Plastics, Lenni, PA) to eliminate any residual R light (<700 nm). Various fluences of dim white light were provided by covering the plates with filter papers (Whatman 3 MM chromatography paper) under the light provided by fluorescent tube (TL70, F17T8/TL741; Philips, Eindhoven, The Netherlands). The green safelight used in the biochemical analysis was obtained by wrapping a green fluorescent tube (GTE Sylvania F40G) with a layer of blue filter (Lee filter HT116; Lee Colotran, Inc., Totowa, NJ) and a layer of green filter (Lee filter 119).
Chlorophyll Assays
Seedlings were ground in darkness in a solution containing 10 mM Tris HCl and 1 mM EDTA, pH 8.0, and chlorophylls were extracted with acetone (1:4, v/v). The amounts of chlorophyll a and b were determined using MacKinney's coefficients (MacKinney, 1941) and the equations chlorophyll a = 12.7 (A663) - 2.69 (A645) and chlorophyll b = 22.9 (A645) - 4.48 (A663).
Scanning Electron Microscopy
Seedlings were fixed with 10% formaldehyde, 10% acetic acid, and 30% ethanol for 1 hr at room temperature and subjected to a dehydration process with ethanol. They were then subjected to a critical drying process and spattered with tungsten particles, and a scanning electron microscope was used to observe the hypocotyl epidermal cells. Seedlings also were observed with the light microscope using differential interference to count hypocotyl cell numbers.
Protein and RNA Gel Blot Analyses
All of the protein gel blot samples were separated on 10% acrylamide gels containing SDS. A polyclonal anti-G antibody used to detect the G protein was raised against a synthetic oligopeptide corresponding to the C-terminal sequence of GPA1, and protein gel blotting was performed according to the method described by Weiss et al. (1993). The primary antibody was probed with peroxidase-conjugated anti-rabbit antibody, and the signal was detected with the enhanced chemiluminescence system (Amersham-Pharmacia). For detection of the loading control, -tubulin antibody (Sigma) was used to hybridize the same membrane used for G detection according to the conditions recommended by the manufacturer.
RNA isolation and RNA gel blot analysis were performed according to our previously described protocols (Torii et al., 1999). The same probes (CAB and RBCS) and labeling method (Torii et al., 1999) were used in this work.
Gel Filtration Chromatography
Seedlings were ground in a buffer containing 30 mM Tris, pH 8.0, 330 mM sucrose, 2.5 mM EDTA, 10% glycerol, 2 mM phenylmethylsulfonyl fluoride, 2 mM DTT, and 1 x complete proteinase inhibitor cocktail (Boehringer Mannheim) and centrifuged at 16,000g at 4°C for 10 min. GTP was added to the supernatant at a final concentration of 10 µM, and samples were centrifuged at 6000g at 4°C for 5 min to further eliminate any precipitated material. Triton X-100 was added to the supernatant at a final concentration of 0.5%, and samples were centrifuged at 120,000g at 4°C for 30 min to remove the microsomal membranes. The supernatant was loaded onto a Superdex 75 gel filtration column (Amersham-Pharmacia). Gel filtration was performed with a buffer containing 50 mM Tris, pH 8, at 4°C, 150 mM NaCl, and 10% glycerol, and protein fractions of 250 µL were collected. Protein in each fraction was concentrated by adding 10 µL of resin (StrataClean; Stratagene), and 2 x SDS sample buffer was added to each of the resins before loading onto acrylamide gels.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSNOTE ADDED IN PROOFReferences |
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Received January 5, 2001; accepted April 20, 2001.
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-Subunit Enhances Phytochrome-Mediated Inhibition of Hypocotyl Elongation in Arabidopsis
