JXB Advance Access originally published online on May 19, 2006
Journal of Experimental Botany 2006 57(9):2101-2110; doi:10.1093/jxb/erj167
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RESEARCH PAPER |
Overexpression of the regulator of G-protein signalling protein enhances ABA-mediated inhibition of root elongation and drought tolerance in Arabidopsis
College of Bioscience and Biotechnology, Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Yangzhou University, Yangzhou 225009, People's Republic of China
To whom correspondence should be addressed. E-mail: jsliang{at}mail.yzu.edu.cn
Received 21 September 2005; Accepted 17 February 2006
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Abstract |
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Regulator of G-protein signalling (RGS) proteins identified recently in Arabidopsis have been involved in the regulation of several physiological processes, but largely nothing is known about their roles at both the physiological and the molecular level. In the experiments reported here, the overexpression approach was used to present evidence that RGS1 protein plays critical roles in plant development and in modulating abscisic acid (ABA) and drought stress signal transduction. RGS1 affected the shapes of leaves, the development of floral buds, the elongation of stems, siliques, and hypocotyls, and the time of flowering. Post-germination growth was inhibited by 1 µM ABA, and root growth was hypersensitive to ABA for 35S-RGS1 transgenic plants. RGS1 overexpression conferred more drought tolerance to transgenic plants, as compared with the wild type (Columbia). Reverse transcription–PCR (RT–PCR) results indicated that RGS1 overexpresssion significantly stimulated the expression of NCED and ABA2, that encode two key enzymes catalysing ABA biosynthesis. Furthermore, the expression of several stress-regulated genes was either up- or down-regulated in RGS1-overexpressing transgenic plants. Combining the results above with previous results, it is suggested that RGS1 exerted its effects on plant responsiveness to ABA and drought tolerance largely through changing the expression either of genes responsible for ABA biosynthesis, which leads to changes in endogenous ABA levels, or of stress-responsive genes.
Key words: ABA, Arabidopsis thaliana, AtRGS1 protein, drought tolerance, RGS1 overexpression
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Introduction |
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Heterotrimeric G-proteins transduce extracellular signals from G-protein-coupled receptors and mediate intracellular processes critical for many cellular processes, such as plant growth responses to hormones, drought, light, and pathogens, and many developmental events (McCudden et al., 2005). The G-protein-mediated signal transduction chain consists of many components, including,
, ß, and 
subunits of the G-protein heterotrimer, G-protein-coupled receptors (GPCRs), regulator of G-protein signalling (RGS) protein, and many other downstream effectors. Once the extracellular signal molecules (i.e. ligands) interact specifically with GPCRs, which results in changes in the conformation of GPCRs, they catalyse the exchange of GDP for GTP on the G subunit of heterotrimeric G-proteins and subsequent dissociation of G from its cognate Gß subunit complex. As a consequence, the activated GTP-bound subunit and ß subunit complex interact separately with a variety of downstream effectors, although little is known about plant G-protein effectors.
Unlike the cases in animals, in which many gene products encoding each subunit of G-protein have been identified, the Arabidopsis genome appears to contain only one putative gene that encodes GPCR protein (Pandey and Assmann, 2004), one gene that encodes a canonical G subunit (Ma et al., 1990), one gene that encodes a Gß subunit (Weiss et al., 1994), and two genes that encode G subunits (Mason and Botella, 2000, 2001). Nevertheless, a number of genetic and pharmacological studies, especially the identification and mutation of genes that encode specific G-protein signalling components in several plant species, have shown that G-protein-mediated signalling plays important roles in a wide range of plant processes, including seed germination and seedling growth responses to light, phytohormones, ozone, sugars, pathogen resistance, etc. (see reviews by Jones and Assmann, 2004; Perfus-Barbeoch et al., 2004).
Although significant differences exist in G-protein components between plants and animals, plants do use similar mechanisms to regulate G-protein-mediated signalling. One of these mechanisms is via the RGS protein (RGS1) pathway, which was recently identified in the Arabidopsis genome (called AtRGS1) (Chen et al., 2003). This protein, unlike all other known RGS proteins, has a predicted seven transmembrane (7TM)-spanning domain structure in the N-terminus, which is similar to a GPCR, as well as an RGS box in the C-terminus which has GTPase-accelerating activity. Thus, one of its functions in the regulation of G-protein signalling is to accelerate the conversion of the active G-GTP-bound form to the inactive G-GDP-bound form, as a consequence desensitizing the G-protein-mediated signalling (Chen et al., 2003). Recently, several studies have shown that AtRGS1 protein may be involved in the regulation of cytokinesis, proliferation of some cell types (Chen et al., 2003), and the responses of seedling development to sugars (Chen and Jones, 2004). Our results show that AtRGS1 protein was implicated in the responses of seed germination to sugars and abscisic acid (ABA) (Chen et al., 2006). However, our understanding of the roles of AtRGS1 proteins in regulating plant growth and development is still very limited and no further information has been provided.
In the present study, was used the transgenic approach to overexpress AtRGS1 protein, as well as reverse transcription–PCR (RT–PCR) to study its expression in different tissues/organs and its roles in altering ABA sensitivity and in improving drought tolerance of plants.
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Materials and methods |
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Plant material
Both wild-type (Columbia ecotype) and transgenic plants were grown in a controlled environment greenhouse under 16 h light, supplied by cool-white fluorescent bulbs (100 µmol m–2 s–1), and 8 h of darkness, at a temperature of 22 °C. For flowering time determination, the plants were grown under continuous white light.
Vector construction
The open reading frame of AtRGS1 was amplified by PCR (primers: 5'-CACCATGGCGAGTGGATGTGCT-3', 5'-ACTCCTTAACCGGGACTACTGCATC-3') from a cDNA library made from seedlings grown in light for 2 weeks and cloned into the pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA, USA), and subcloned into Gateway plant transformation destination vector pGWB2 (for plant transformation) (Research Institute of Molecular Genetics, Matsue, Japan) and pK7FWG2 (for cell transformation and protoplast transfection) (Howard Hughes Medical Institute, San Diego, CA, USA), respectively, by an LR recombination reaction. In the above two vectors, expression of RGS1 was driven by the cauliflower mosaic virus (CaMV) 35S promoter.
Arabidopsis transformation
After the LR reaction, the plasmids were transformed into Arabidopsis thaliana (Col-0 ecotype) according to the vacuum infiltration method (Bechtold and Pelletier, 1998), using Agrobacterium tumefaciens strain GV3101. Transgenic seeds were screened on growth medium-Suc plates containing 100 µg ml–1 kanamycin and 100 µg ml–1 hygromycin B. The surviving plants were combined into pools of 10 plants and grown in soil under greenhouse conditions. For the phenotype analysis, only one T3 homozygous line with high RGS1 expression was used.
Cell transformation
Wild-type cells were maintained in Murashige and Skoog (MS) liquid medium containing 0.2 µg ml–1 2,4-D at 24 °C with shaking at 150 rpm on a gyratory shaker and subcultured every week with a 5% inoculum. Three days after subculture, 4 ml of the plant cells were transferred to 100 mm Petri dishes and incubated at 24 °C with 100 µl of fresh culture of A. tumefaciens containing the vectors above, the bacterial cells were washed off and the plant cells were plated on the MS agar medium containing 0.2 mg ml–1 2,4-D, 500 µg ml–1 carbenicillin, and various amounts of spectinomycin. The spectinomycin-resistant transformants were selected 2–3 weeks after screening.
Protoplast transfection and analysis of intracellular localization of RGS1 protein
The transfection experiments were conducted on protoplasts isolated from 3-week-old Arabidopsis. About 20–30 leaves were cut in 1 mm leaf strips and put into 5 ml enzyme solutions: 0.4 M mannitol, 20 mM KCl, 20 mM MES, 10 mM CaCl2, 5 mM ß-mercaptoethanol, 0.1% bovine serum albumin (BSA), pH 5.7, containing 1% cellulase R-10 and 0.2% macerozyme R-10 (Yakult Honsha, Tokyo, Japan). The Petri dishes containing the enzyme solution and leaf strips were covered with aluminium foil and subjected to a vacuum for 30 min, and then incubated for another 3 h in the dark with shaking at 40 rpm (25 °C). The protoplasts were harvested after 600 g centrifugation for 2 min, washed with 6 ml of washing solution containing 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES, pH 5.7, and re-suspended with the same solution (the protoplast density was 
2x105 cell ml–1). After being kept on ice for 30 min, the protoplasts were pooled after centrifugation and re-suspended in a solution containing 0.4 M mannitol, 15 mM MgCl2, 4 mM MES, pH 5.7.
For protoplast transfection, 20 µg of DNA from the constructed vectors, 200 µl of protoplast suspension, and 200 µl of 40% polyethylene glycol (PEG) 4000, 0.1 M Ca(NO3)2, and 0.4 M mannitol, pH 8.0, were mixed by gentle inversion at room temperature for 15 min. After washing with washing solution, the transfected protoplasts were re-suspended in 200 µl of solution containing 0.5 M mannitol, 20 mM KCl, 4 mM MES-KOH, pH 5.7, at room temperature in the dark.
For analysis of the intracellular localization of RGS1–green fluorescent protein (GFP), the transformed cells and transfected protoplasts were transferred to 5% calf serum-coated 12-well plates and observed with a fluorescence microscope (Olympus BX 51TRF).
ABA treatment
Sterilized seeds were stratified at 4 °C for 48 h. For root length determination, seeds were germinated on an ABA-free medium for 3 d and 10 uniform and well-grown seedlings were carefully transferred to media containing various concentrations of ABA. Root elongation was measured 5 d after transplantation.
Drought-stress treatment and water-loss analysis
Seeds of 35S-RGS1 and the wild type were germinated as normal. Seedlings with 3–4 leaves were transplanted to soil in the same tray and grown under normal conditions in a greenhouse (16 h day/8 h night, 22 °C). Soil drying treatments were imposed by withholding water when plants grew to 8–10 leaves. After 15 d of drought treatment, drought-stressed plants were rewatered.
The water loss of detached leaves was measured by weighing the leaf at a specified time. Ten fully expanded leaves were harvested and placed on open Petri dishes under strictly controlled conditions.
Stomatal aperture bioassays
Stomatal aperture bioassays using isolated abaxial epidermal peels from fully expanded leaves of wild-type and 35S-RGS1 transgenic plants were conducted as described by Wang et al. (2001). Epidermal strips were pretreated with buffer for 3 h and apertures of opened stomata were measured after a subsequent incubation in buffer containing various concentrations of ABA for different incubation times.
RNA isolation and transcript abundance analysis by RT–PCR
Total RNA for RT–PCR was prepared from plants by using the RNeasy Plant Minikit (Qiagen). RNA was used as a template for first-strand DNA synthesis using the SuperScript First-Strand Synthesis System (Invitrogen Life Technologies). First-strand cDNA was used for PCR amplification. PCR was carried out using Taq DNA polymerase (Invitrogen), and gene-specific primers used were as follows: rd29A, 5'-atcacttggctccactgttgttc-3' and 5'-acaaaacacacataaacatccaaagt-3'; ICK1, 5'-ccgtcgtcggtgataatgga-3' and 5'-ctaatggcttctccttctcg-3'; ABI1, 5'-tcaagattccgagaacggagatc-3' and 5'-gaggatcaaaccgaccatctaac-3'; ABI2, 5'-gttcttgttctggcgacggagc-3' and 5'-ccattagtgactcgaccatcaag-3'; ABA2, 5'-aaagtggcattgatcact-3' and 5'-tcctagtcaagcctaga-3'; NCED3 5'-cgtgaaatccgtacggaacc-3' and 5'-ccggaatccggtgaactctt-3'; SUS1, 5'-tcgggacgaatctgttgag-3' and 5'-gattcgatgtgatggcaagcac-3'; ADH1, 5'-tccacgtatcttcggccatg-3' and 5'-tagcaccttctgcagcgcc-3'; CHS, 5'-tcaccaacagtgaacacatgacc-3' and 5'-gagtcaaggtgggtgtcagagg-3'; and ACTIN, 5'-tgggatgacatggagaagat-3' and 5'-ataccaatcatagatggctgg-3'.
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Results |
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AtRGS1 is expressed in all different organs
The functions of genes can be explored in several ways. One of the most powerful approaches is to isolate and characterize mutations of the genes that encode specific proteins and to compare the differences in the critical processes for plant growth and development between mutants and their wild types. Another useful technique is to construct overexpression lines of the genes under investigation. In the present study, AtRGS1-overexpressing lines were constructed and the roles of AtRGS1 investigated by comparing the differences in the responses to ABA and drought between AtRGS1-overexpressing lines and the wild type. The coding region of AtRGS1 was fused to the 35S promoter of CaMV and the construct was transformed into A. thaliana (Col-0) by Agrobacterium-mediated transformation. Only the transgenic lines with the highest RGS1 expression levels were selected for more detailed analysis.
Using RT–PCR, the expression patterns of RGS1 were analysed and it was found that RGS1 was expressed in all the different plant organs tested. However, the expression levels of RGS1 were quite different, and a higher expression level was observed in leaves and roots and a lower expression level in flower buds, stems, and siliques. In transgenic plants, RGS1 expression was strongly stimulated in various organs, especially in leaves, stems, and roots, as compared with that in the wild type (Fig. 1).
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Growth is retarded in AtRGS1overexpression line
The differences in phenotype between wild-type and 35S-RGS1 transgenic plants were analysed. Compared with the wild-type plants, 35S-RGS1 transgenic plants exhibited obvious growth retardation in the aerial parts; shorter plant height and petioles were observed, but no significant differences in phenotype of siliques and rosettes were detected. Furthermore, the floral buds at the inflorescence apex appear more tightly dispersed than they were in the wild type. The hypocotyls were also shorter in 35S-RGS1 plants when grown in the dark for 2 days (Fig. 2).
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The time needed for the transition from vegetative to reproductive growth in 35S-RGS1 transgenic and wild-type plants was recorded. Time to flowering was determined by counting the numbers of days after stratification until the day the first flower opened. Under long-day growth conditions, which are known to induce flowering in Arabidopsis plants, 50% of wild-type plants were flowering in <30 d; however, only 15% of 35S-RGS1 transgenic plants were flowering during the same period (Fig. 3), implying that RGS1 proteins were involved in the regulation of plant development.
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35S-RGS1 plants are hypersensitive to ABA
A number of studies have shown that the plant hormone ABA plays central roles in many physiological processes, especially during stresses (Finkelstein and Rock, 2002). ABA, as a stress hormone, mediates the plant's response to drought by its rapid accumulation, thus helping plant survival (Zhang et al., 2004). Our previous studies showed that overexpression of RGS1 obviously stimulated ABA biosynthesis (Chen et al., 2006), suggesting that RGS1-mediated signalling may be related to ABA action. Here, it is shown that RGS1 overexpression significantly affected a plant's responses to ABA. In addition to altering the sensitivity of seed germination to ABA (Chen et al., 2006), the cotyledon opening and greening of 35S-RGS1 seedlings were also significantly delayed by 1 µM ABA and almost all cotyledons failed to open and to become green, as compared with those with no ABA treatment. Fewer effects of ABA on cotyledon opening and greening were observed in Col plants (Fig. 4A, B). Significant differences in ABA inhibition of root growth were also detected between wild-type and transgenic seedlings. When seedlings were grown in medium without ABA, the root growth of 35S-RGS1 seedlings was almost the same as that of the wild type; however, when seedlings were transferred from growth medium without ABA (control) to medium supplemented with different ABA concentrations from 0 to 10 µM, root growth of the two genotypes was markedly inhibited as the ABA concentration increased, and much more significant inhibition was observed in 35S-RGS1 seedlings. Furthermore, even in the media with higher ABA concentrations (from 5 to 10 µM ABA), the roots of the wild type still retained a relatively higher growth rate, whereas the root growth of 35S-RGS1 seedlings was almost completely inhibited at the same ABA concentration (Fig. 4C, D).
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35S-RGS1 transgenic plants exhibit more drought tolerance
To determine whether RGS1 protein is involved in the regulation of Arabidopsis tolerance to drought, soil drying treatments were imposed at the stage of 8–10 fully expanded leaves by withholding water from soil-grown plants. As shown in Fig. 5A, when plants were subjected to drought stress for 15 d, the leaves of the wild type displayed severe withering and became yellow in colour, whereas most leaves of 35S-RGS1 transgenic Arabidopsis still retained turgor and were healthy. Upon rewatering, the growth of leaves of 35S-RGS1 transgenic plants rapidly recovered and the plants displayed normal flowering, whereas only a few wild-type plants survived to maturity. These results suggested that RGS1 proteins were involved in the regulation of the drought response, although its mechanisms were still unknown.
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The enhanced drought tolerance of the 35S-RGS1 plants could result, at least in part, from its lower transpiration rates. This hypothesis was verified by comparing the water loss rate of detached leaves between the two genotypes investigated. When fully expanded rosette leaves of both genotypes were detached and exposed to controlled conditions, the water loss from detached 35S-RGS1 leaves was significantly lower than that from wild-type leaves. After 240 min, the relative water loss of 35S-RGS1 leaves was 29.8%, whereas a water loss as high as 50.8% was detected for wild-type leaves (Fig. 5B). Stomatal characterization of both genotypes with a scanning electron microscope showed that 35S-RGS1 leaves had a lower stomatal density and smaller aperture (data not shown). Furthermore, ABA-induced stomatal closure was more rapid and significant in 35S-RGS1 leaves than in wild-type leaves (Fig. 5C).
The intracellular localization of RGS1 protein is affected by ABA
Figure 6 shows the intracellular localization of RGS1 protein and the effects of ABA on RGS1 localization in both protoplasts and cells. The results indicated that non-fused GFP proteins appeared to be distributed equally to the whole protoplast, whereas RGS1–GFP fusion proteins were mainly targeted to the cell membrane after 16 h of transfection (Fig. 6A, B), which was consistent with the results reported by Chen et al. (2003). However, when the protoplasts were incubated in medium supplemented with 10 µM ABA for another 16 h, the expression of RGS1 was strongly induced and some RGS1 proteins were moved laterally along the cell membrane and clustered in some regions of the cell membrane, but a large amount of RGS1 proteins was targeted to the nucleus (Fig. 6C). Similar results were also observed with cells (Fig. 6D, E, F). It was still unclear how ABA affected the localization of RGS1 protein and what was the physiological significance of the change of RGS1 protein localization in the ABA signal transduction pathway.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In metazoans, heterotrimeric G-proteins couple stimulus perception by GPCRs with numerous downstream effectors. RGS proteins act as an effective desensitizer of G-protein-mediated signalling by accelerating the deactivation of G-protein
subunits from the G-protein-GTP binding form to the G-protein-GDP binding form. RGS proteins are involved in a variety of cellular functions (McCudden et al., 2005) but, in plants, the functions of RGS1 proteins are largely unknown. It appears that the Arabidopsis genome contains only one member of the RGS proteins, AtRGS1 (Chen et al., 2003). AtRGS1 has a high amino acid sequence similarity to RGS proteins in other eukaryotic organisms, and is composed of a predicated N-terminal 7TM domain and a C-terminal RGS box. Genetic and biochemical evidence shows that AtRGS1 strongly interacts with the G subunit (AtGPA1) and has a GTPase activity, implying that AtRGS1 protein may be involved in G-protein-mediated signalling (Chen et al., 2003). Further studies have shown that AtRGS1 modulates plant cell proliferation and is involved in sugar signalling (Chen and Jones, 2004; Chen et al., 2006). However, many aspects of the roles of AtRGS1 protein in regulating physiological processes in plants are still to be elucidated.
In these experiments, the overexpression approach was used to present evidence that RGS1 protein played critical roles in plant development and in modulating ABA and drought stress signal transduction. RGS1 affected the shapes of leaves, the development of floral buds, and the elongation of stems, siliques, and hypocotyls. Some aspects shared the same phenotypes exhibited by loss of function of the G-protein subunit, as reported previously (Ullah et al., 2001, 2003; Jones et al., 2003), which was consistent with the basic roles of RGS1 protein as a desensitizer of G-protein-mediated signalling. 35S-RGS1 transgenic plants exhibited a moderate late-flowering phenotype when grown under long-day conditions. However, there was no difference in flowering time between 35S-RGS1 transgenic plants and gpa1 mutants (Lapik and Kaufman, 2003; unpublished data), suggesting that RGS1-mediated flowering is a G-protein-independent process.
Many data have shown that ABA plays important roles both in non-turgor-dependent growth inhibition and in stress-dependent processes (Zhang et al., 2004). Recent studies have revealed that ABA is involved in many G-protein-mediated processes (Wang et al., 2001; Chen et al., 2006). It is reasonable to assume that there is a close interaction between RGS1 protein-mediated signalling and ABA signalling, because RGS1 functions as a G subunit-accelerating protein (GAP), although the exact mechanisms were unknown. Based on our previous results using the null transcript mutant of RGS1 (rgs1-2), RGS1 is involved in the regulation not only of ABA biosynthesis, but of cell sensitivity to ABA (Chen et al., 2006). Here, further evidence is provided that RGS1-overexpressing transgenic lines exhibited a hypersensitivity to ABA both in seedling cotyledon opening and root growth (Fig. 4), and in stomatal closing (Fig. 5C), which was consistent with our previous results showing that the seed germination of the rgs1-2 mutant was insensitive to ABA (Chen et al., 2006). However, based on the response of stomatal opening of the gpa1 mutant to ABA, i.e. that ABA treatment did not inhibit its stomatal opening (Wang et al., 2001), it would be expected that the AtRGS1-overexpressing line is also less sensitive to ABA signalling in Arabidopsis guard cells, since AtRGS1 is thought to accelerate the conversion from the active G-ATP-binding form to the inactive G-ADP-binding form. These opposite results could be explained by the fact either that different experimental systems were used or that AtRGS1 has other functions in addition to its role as a GAP. Nevertheless, these results and our previous results implied that the many RGS1-mediated physiological processes were, at least in part, mediated via the control of ABA biosynthesis and/or changes in plant cell sensitivity to ABA. The expression patterns of RGS1 in different plant organs were, at least in part, consistent with their functions (Fig. 3).
It is well known that ABA, as a stress hormone, is involved in mediating the plant drought response (Zhang et al., 2004). This specific role has been extensively investigated and abundant information has been accumulated over the past two decades that ABA can act as a long-distance water stress signal in sensing soil drying. The drought stress-induced ABA in dehydrated roots in drying soil is transported through a transpiring flux of xylem to adjacent guard cells and induces stomatal closure and inhibits leaf growth, and, as a consequence, decreases the water loss by transpiration and maintains cell turgor. Understandably, therefore, if RGS1 was involved in the control of ABA biosynthesis and of cell sensitivity to ABA, it could also be expected to regulate the plant response to soil drying. As expected, RGS1-overexpressing plants were much more tolerant to soil desiccation than the wild type (Fig. 5A).
As mentioned above, the effects of RGS1 on plant growth and development, and tolerance to drought stress were largely through changing endogenous ABA biosynthesis and/or ABA responsiveness. Therefore, it is reasonable to assume that RGS1 can exert its effects on expression of genes responsible either for ABA biosynthesis or for ABA responsiveness. In higher plants, it has been well documented that the biosynthesis of ABA is through an indirect pathway (Taylor et al., 2000; Xiong and Zhu, 2003). Two crucial enzymes, 9'-cis-epoxycarotenoid dioxygenase (encoded by NCED), which catalyses the oxidative cleavage from 9'-cis-neoxanthin to xanthoxin, and xanthoxin oxidase (encoded by ABA2), which catalyses xanthoxin oxidation to form AB-aldehyde, are considered to be key regulators (Schwartz et al., 1997; Iuchi et al., 2001). It has been shown that the expression of these two genes is upregulated by water stress. Our previous studies indicated that their expression in a null mutant of RGS1 was inhibited under either normal conditions (non-stress) or osmotic stress conditions, which can explain why the rgs1-2 mutants lacked the osmotic stress-induced ABA accumulation (Chen et al., 2006). This hypothesis was further verified by the RT–PCR results which indicated that overexpression of RGS1 significantly increased the transcript abundance of ABA2 and NCED3 (Fig. 7).
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Recently, analyses on gene expression profiling using cDNA microarrays or gene chips have identified in Arabidopsis that the expression of >200 genes is regulated by ABA, and some of these are functional genes (Seki et al., 2002). Here, the expression of several well-known ABA-regulated genes in 35S-RGS1 plants were also analysed using RT–PCR, and the results showed that the transcriptional levels of rd29A, which is up-regulated by ABA and abiotic stresses (Yamaguchi-Shinozaki and Shinozaki, 1994), and of ICK1, which mediates cell division arrested by ABA (Wang et al., 1998), were much higher in 35S-RGS1 plants than in the wild type. The expression of ABI1 and ABI2 that encode similar class 2C serine/threonine protein phosphatases (Meyer et al., 1994; Rodriguez et al., 1998), was slightly higher than that of the wild type, whereas the expression of SUS1 (Martin et al., 1993), ADH1 (de Bruxelles et al., 1996), and CHS (Feinbaum and Ausubel, 1988) was down-regulated in 35S-RGS1 plants. These results suggested that RGS1 is involved in the regulation of gene expression and acted as either a positive or a negative regulator in ABA and water-stress signalling.
Increasing evidence has shown that there is a close relationship between protein function and its intercellular location. Prototypical RGS proteins are first recognized in genetic screens of lower eukaryotes as negative regulators of G-protein signalling (Siderovski et al., 1996), but growing evidence indicates that many family members perform additional cellular tasks (Hepler, 1999). In metazoans, RGS proteins have a variety of domains for protein–protein interaction, in addition to the RGS domain (Hollinger and Hepler, 2002), and thus selectivity for activation of particular pathways may be obtained by scaffolding mechanisms linking RGS protein to particular G proteins and/or receptor proteins and signalling pathways. In Arabidopsis, the open reading frame of AtRGS1 consists of 459 amino acids, which contains an RGS box in the C-terminus with 211 amino acids and a 7TM domain in the N-terminus (Chen et al., 2003). So, theoretically, this protein belongs to the family of membrane proteins and localizes in the membrane, which was also experimentally verified in the present study using the GFP–RGS1 fusion technique (Fig. 6B; Chen et al., 2003). Unexpectedly, the subcellular localization of RGS1 protein can be changed by ABA treatment both in protoplasts and in cells, (Fig. 6C, F). At present, nothing is known about the mechanisms and significance of RGS1 protein targeting, but this is a very interesting topic and is worthy of further study.
To summarize, based on the results presented herein, concluded that RGS1 regulates many plant physiological processes and exerted its roles largely through changing ABA biosynthesis and ABA responsiveness (Fig. 8). Overexpression of RGS1 enhanced plant sensitivity to ABA and tolerance to drought.
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Acknowledgements |
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This work was supported by the National Science Foundation of China (grant no. 30370731) and State Key Basic Research and Development Program of China (grant no. 2003CB114303).
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Footnotes |
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* These authors contributed equally to this work.
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Abbreviations |
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ABA, abscisic acid; CaMV, cauliflower mosaic virus; GFP, green fluorescent protein; GPCR, G-protein-coupled receptor; RGS, regulator of G-protein signalling; RT–PCR, reverse transcription–PCR.
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