JXB Advance Access originally published online on July 7, 2006
Journal of Experimental Botany 2006 57(11):2697-2708; doi:10.1093/jxb/erl035
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RESEARCH PAPER |
RACK1 mediates multiple hormone responsiveness and developmental processes in Arabidopsis
1Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, British Columbia, V6T 1Z4 Canada
2Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
3Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
4Structural BioInformatics Core Facility, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
5Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA
6College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, PR China
*To whom correspondence should be addressed. E-mail: jingui{at}interchange.ubc.ca
Received 26 January 2006; Accepted 20 April 2006
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Abstract |
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The scaffold protein RACK1 (Receptor for Activated C Kinase 1) serves as an integrative point for diverse signal transduction pathways. The Arabidopsis genome contains three RACK1 orthologues, however, little is known about their functions. It is reported here that one member of this gene family, RACK1A, previously identified as the Arabidopsis homologue of the tobacco arcA gene, mediates hormone responses and plays a regulatory role in multiple developmental processes. RACK1A expresses ubiquitously in Arabidopsis. Loss-of-function mutations in RACK1A confer defects in multiple developmental processes including seed germination, leaf production, and flowering. rack1a mutants displayed reduced sensitivity to gibberellin and brassinosteroid in seed germination, hypersensitivity to abscisic acid in seed germination and early seedling development, and hyposensitivity to auxin in adventitious and lateral root formation. These results provide the first genetic evidence that RACK1A is involved in multiple signal transduction pathways.
Key words: Abscisic acid, auxin, brassinosteroid, flowering, gibberellin, RACK1, seed germination, signal transduction
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Signal molecules, such as plant hormones, regulate diverse processes in plant growth and development. Classic genetic screens have yielded various components involved in signal transduction. Recently, microarray-based analyses have proven to be another productive approach for the discovery of genes in plant hormone signal integration. From these studies, it is now clear that numerous genes can be regulated by a single signal, such as auxin (Pufky et al., 2003). The reciprocal is true; a single gene can be regulated by many different biological or environmental stimuli. For example, some genes originally defined as auxin-inducible genes can also be induced by other plant hormones, such as brassinosteroids (Goda et al., 2004; Nemhauser et al., 2004). It has been recognized for many years that signals integrate and the term ‘cross-talk’ was coined to describe this complexity (see recent reviews by Gazzarrini and McCourt, 2003; Chinnusamy et al., 2004). However, it remains largely unclear how signalling components interact with each other, and how signals are integrated.
One mechanism by which eukaryotic cells are able to integrate multiple signals is through the organized proximity of signalling components using scaffold proteins. Scaffold proteins assemble active complexes with multiple signalling proteins/enzymes from the same or different pathways to facilitate the interaction and regulation of signal transduction networks. For a growing number of signalling pathways, scaffold proteins are shown to be central elements. For example, the yeast scaffold protein, Ste5p, simultaneously binds different members of the mitogen-activated protein kinase (MAPK) cascade to transduce signals controlling mating (Harris et al., 2001; Park et al., 2003). This same scaffold is used in the osmolarity response but concatenating a different set of MAPK kinases (Park et al., 2003).
The ß propeller motif has emerged as a common platform for integrating various signals. Beta propeller proteins are diverse (van Nocker and Ludwig, 2003); one member of this large family of proteins is designated Receptor for Activated C Kinase 1 (RACK1). Mammalian RACK1 has a seven-bladed propeller structure with one Trp-Asp 40 (WD40) unit constituting each blade. The WD40 repeat is thought to be involved in protein–protein interactions, and the ß propeller structural unit may enable the protein to form reversible complexes with its ligand proteins. Increasing evidence suggests that interaction domains, such as ß propeller, have been evolutionarily selected for their flexibility, their ability to assemble multiprotein machines, and their potential to mediate sophisticated biological functions (Fulop and Jones, 1999; Pawson and Nash, 2003). Originally identified as an anchoring protein for protein kinase C (PKC) in mammals (Ron et al., 1994) which shuttles the active enzyme to different cellular sites, RACK1 is now viewed as a versatile scaffold protein, that can bind numerous signalling molecules from diverse signal transduction pathways in a regulated fashion (see review by McCahill et al., 2002). Some recent examples include cAMP signalling (Yarwood et al., 1999; Steele et al., 2001), cell cycle control (Mamidipudi et al., 2004), Ca2+ release (Patterson et al., 2004), ribosome assembly and mRNA translation regulation (Ceci et al., 2003; Nilsson et al., 2004; Sengupta et al., 2004), cytoskeleton remodelling (Osmanagic-Myers and Wiche, 2004), and proteasome degradation (Fomenkov et al., 2004).
RACK1 is a highly conserved protein found in both animals and plants. Positioning of RACK1 WD repeats is conserved in the alga Chlamydomonas reinhardtii which diverged from the forerunners of the plant kingdoms about 1 billion years ago, indicating that the biological function of RACK1 is ancient (Neer et al., 1994; McCahill et al., 2002). Although not recognized as such, the first RACK1 homologue in plants was identified in tobacco BY-2 suspension cells as an auxin-inducible gene, arcA (Ishida et al., 1993), and later in Arabidopsis, rice, rape, and alfalfa (Iwasaki et al., 1995; McKhann et al., 1997; Vahlkamp and Palme, 1997; Kwak et al., 1997). Indirect evidence suggested that plant RACK1 may be involved in hormone-mediated cell division, UV and salicylic acid responses (Ishida et al., 1993; McKhann et al., 1997; Perennes et al., 1999). However, direct genetic evidence has been lacking, thus no signal transduction pathway involving a RACK1 has been unequivocally identified in plants. The first genetic evidence that Arabidopsis RACK1 has a role in hormonal signalling pathways and may have a regulatory role in multiple developmental processes is provided here.
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Materials and methods |
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Plant materials
Initially, deconvoluted pools of DNA from T-DNA transformed plants were used to screen for insertions in the Arabidopsis RACK1A gene (At1g18080). A total of 60 000 T-DNA insertion lines in the Columbia (Col-0) ecotype (Alonso et al., 2003) were screened by PCR using RACK1A- and T-DNA-specific primers. A putative insertion line was identified by using primers specific for the RACK1A 5'-UTR (5'-GGCATCTCCAGACACCGAAA-3') or 3'-UTR (5'-GCAGAGAGCAACGACAGC-3') together with a T-DNA left border-specific primer (5'-GGCAATCAGCTGTTGCCCGTCTCACTGGTG-3'). A single insertion in the first exon of RACK1A was isolated, and the insertion was confirmed by sequencing. This rack1a mutant allele was designated rack1a-1. The second rack1a mutant allele (rack1a-2, SALK_073786) was obtained from the Salk Institute sequence-indexed insertion mutant collection (http://signal.salk.edu/cgi-bin/tdnaexpress). The T-DNA insertion site in this allele was at the first intron of RACK1A. Plants homozygous for rack1a-2 were isolated, and the insertion was confirmed by sequencing. Loss of detectable RACK1A transcripts in rack1a-1 and rack1a-2 mutants was verified by RT-PCR. Total RNA was isolated from 10-d-old, light-grown seedlings by using the TRIzol reagent (Life Technologies). cDNA was synthesized using 1 µg of total RNA by Oligo(dT)20-primed reverse transcription, using THERMOSCRIPT RT (Life Technologies). RACK1A primers described above that flank the insertion sites and Arabidopsis ABP1 (At4g02980) primers (5'-TGATCGTACTTTCTGTTGGTTCC-3' and 5'-CCAATAGTAAGGGAACTTCAGCC-3' were added together in each RT-PCR reaction. All mutant alleles are in Col-0 ecotype.
Molecular modelling
The sequence of RACK1A was submitted to 4 fold-recognition web servers to identify structural templates for homology modelling. The fold-recognition servers were bioinbgu (www.cs.bgu.ac.il/
bioinbgu/), 3D-PSSM (www.sbg.bio.ic.ac.uk/3dpssm/; Kelley et al., 2000), GenTHREADER (bioinf.cs.ucl.ac.uk/psipred/index.html; Jones, 1999), and FUGUE (www-cryst.bioc.cam.ac.uk/fugue/prfsearch.html; Shi et al., 2001). The fold-recognition servers identified Gß1 (PDB ID 1GOT
[PDB]
) from the Protein Data Bank (Berman et al., 2000) as a template for modelling RACK1A. A model of RACK1A and human RACK1 was built using the modeller module of the InsightII molecular modelling system from Accelrys Inc. (www.accelrys.com). The homology model was evaluated for sequence-structure compatibility using the verify function of the Profiles-3D module from InsightII. The theoretical model for RACK1A was given a self-compatibility score of 150.2 by the Profiles-3D/Verify module of InsightII. The typical score expected for an experimentally-determined protein structure of 323 residues was 147.1, indicating the model is plausible. Similar results were obtained for the model of human RACK1 with a self-compatibility score of 135.8 and a typical score of 141.8 for a 311 residue globular protein. For comparison, the self-compatibility score for the 340 residue mammalian Gß1 structural template was 165.9, with a typical score of 154.4. Figures were created using PyMOL (http://pymol.sourceforge.net/).
Germination assay
The procedure of germination assay was according to Chen et al. (2004). Briefly, sterilized wild-type and mutant seeds from matched lots were pretreated with 8 µM paclobutrazol (Chem Service, West Chester, PA) in the dark at 4 °C for 2 d, washed six times with deionized water, sown on MS/G plates consisting of 
Murashige and Skoog (MS) basal medium with vitamins (plantmedia, Dublin, Ohio), 1% (w/v) sucrose, 0.5% (w/v) phytoagar (plantmedia, Dublin, Ohio), pH adjusted to 5.7 with 1 N KOH, and with different concentrations of gibberellic acid (GA3) or brassinolide (BL). The plates were pretreated with 80 µmol m–2 s–1 light for 6 h. After 3 d in darkness at 23 °C, the percentage of germination was scored. Germination was defined as an obvious protrusion of the radicle through the seed coat. Each experiment was repeated three times. Minimally 50 seeds were scored for each treatment of each genotype.
For the ABA assay, sterilized wild-type and mutant seeds from matched lots were directly sown on MS/G plates with different concentrations of ABA, and cold-treated at 4 °C in the dark for 2 d. Two days after the plates were moved to 23 °C, with 14/10 h photoperiod at 120 µmol m–2 s–1, the percentage of seed germination was scored. Another 3 d later, the percentage of green seedlings was measured. Each experiment was performed with three replicates.
Root assays
The assay for auxin-induced lateral root formation was according to the protocol of Ullah et al. (2003) with modifications. Seedlings were grown on MS/G plates with the addition of 5 µM naphthylphthalamic acid (NPA), an auxin polar transport inhibitor, for 9 d. The NPA-pretreated seedlings were then transferred to MS/G plates with or without 0.1 µM 1-naphthaleneacetic acid (NAA, plantmedia, Dublin, Ohio), and grown vertically under a 14/10 h photoperiod at 120 µmol m–2 s–1. The numbers of lateral roots per length of primary root were scored 4 d later. The standard error of the mean was calculated based on at least 10 seedlings.
The assay for auxin-induced adventitious root formation was according to the protocol of Kubo and Kakimoto (2000) with modifications. Seedlings were grown for 6 d in dim light (5 µmol m–2 s–1). Hypocotyls were excised aseptically and transferred to Petri dishes containing 1x MS basal medium with Gamborg's vitamins, 1% (w/v) sucrose, 0.3% (w/v) phytoagar, pH adjusted to 5.7 with 1 N KOH, with or without 0.5 µM of 1-NAA. Excised hypocotyls were grown for additional 14 d under a 14/10 h photoperiod at 80 µmol m–2 s–1. The number of adventitious roots per length of hypocotyl was scored under a dissecting microscope. The standard error of the mean was based on at least 10 excised hypocotyls.
Yeast split-ubiquitin assay
Interaction between RACK1A and GPA1 was assessed by complementation of split ubiquitin domain fusion in yeast (Fetchko and Stagljar, 2004). The entire open-reading frame of RACK1A was cloned and fused in frame to the C-terminal domain of ubiquitin in pMet AtMlo1 Cub R-Ura3 bait vector (Kim et al., 2002), kindly provided by Dr Ralph Panstruga (Max-Planck-Institüt für Züchtungsforschung-Köln, Germany). The construction of GPA1 prey vector, host strain used, yeast transformation and selection, and identification of interaction have been described previously (Chen et al., 2003). Interaction between RACK1A and GPA1 was analysed based on yeast growth in selective media containing 5-fluoro-orotic acid. The MLO1-calmodulin interaction (pAtMlo1 NuI-Hv CaM3) (Kim et al., 2002) was used as positive controls, and the point mutant MLO1 (pAtMlo1 NuG)-calmodulin interaction was used as a negative control (Kim et al., 2002).
RACK1 promoter:ß-glucuronidase (GUS) constructs
Genomic DNA 2740 bp upstream of the RACK1A start codon, 2215 bp upstream of the RACK1B start codon, and 1091 bp upstream of the RACK1C start codon was amplified by PCR, and cloned into the pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA) respectively, then moved into the Gateway plant transformation destination vector pBGWFS7 (Karimi et al., 2002) by LR recombination reactions. The binary vector was transformed into Arabidopsis thaliana (Col-0 ecotype) by Agrobacterium-mediated transformation (Bechtold and Pelletier, 1998). There are 4113 bp, 43 bp, and 371 bp separating RACK1A, RACK1B, and RACK1C genes from their nearest neighbour genes, respectively. The exact promoter regions for RACK1A, RACK1B, or RACK1C genes could not be determined. However, the regions chosen contain typical promoter-proximal elements such as CAAT box and GC box that are necessary for gene transcription, and probably contain most cis-acting regulatory elements that are important for the regulation of transcription.
Six-day-old whole seedlings and floral organs from transgenic Arabidopsis plants containing RACK1A, RACK1B, or RACK1C promoter:GUS fusion constructs were sampled for GUS histochemical staining. Briefly, seedlings or excised tissues were vacuum infiltrated for 1 min with freshly-prepared staining solution [100 mM Na2HPO4 and NaH2PO4, pH 7.0, 10 mM EDTA, 0.1% (v/v) Triton X-100, 20% (v/v) methanol, and 1 mM 5-bromo-4-chloro-3-indolyl ß-D-glucuronide (X-Gluc; Rose Scientific Ltd., Edmonton, Alberta, Canada)]. After incubation at 37 °C in the dark overnight, seedlings or tissues were cleared in 70% ethanol, examined with a dissecting or compound microscope, and photographed with a digital camera.
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Results |
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Arabidopsis arcA is not a heterotrimeric G-protein ß subunit as previously proposed
The first plant RACK1 orthologue was originally cloned as an auxin-responsive gene, arcA, in tobacco BY-2 suspension cells in a differential screen for genes involved in auxin-mediated cell division (Ishida et al., 1993). Vahlkamp and Palme (1997) further found such an arcA homologue in Arabidopsis. arcA was proposed to be a putative heterotrimeric G-protein ß subunit (Gß) (Ishida et al., 1993), a subgroup of the large family of WD40 repeat proteins (van Nocker and Ludwig, 2003). In order to be consistent with the nomenclatures in recent publications (Chang et al., 2005; Giavalisco et al., 2005), and for clarity hereafter, the Arabidopsis arcA gene (At1g18080), and arcA gene product will be referred to as RACK1A and RACK1A (described below), respectively. Compared with the genuine Arabidopsis Gß, AGB1, RACK1A shares 26% identity and 43% similarity (Fig. 1A). Molecular modelling data revealed that both Gß and RACK1A have a very similar circular seven-bladed propeller structure (Fig. 2). However, RACK1A lacks the N-terminal helix of Gß which is critical for G
interaction (Fig. 2). Therefore, although RACK1A is similar to Gß in structure, it is unlikely that it can function as a Gß.
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This was tested further by analysing the interaction of RACK1A and the sole Arabidopsis heterotrimeric G-protein
subunit, GPA1, in a yeast split-ubiquitin assay. The assay that was established to test the interaction between GPA1 and AtRGS1, the sole Regulator of G-protein Signalling in Arabidopsis (Chen et al., 2003) was used. GPA1 and RACK1A were cloned into the N- and C-terminal domain of ubiquitin vector, respectively. As a control, the sole Arabidopsis heterotrimeric G-protein ß subunit, AGB1, was used. The interaction was analysed based on yeast growth in selective media containing 5-fluoro-orotic acid. As shown in Fig. 3, AGB1 interacts with GPA1 whereas RACK1A does not interact in the yeast split-ubiquitin assay. Because RACK1A does not bind the sole Arabidopsis heterotrimeric G-protein subunit in this configuration (Fig. 3), and because RACK1A lacks the critical N-terminal helix of Gß required for binding to G (Fig. 2), it was concluded that RACK1A is not a heterotrimeric G-protein ß subunit.
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RACK1 orthologues in Arabidopsis
The BLAST search of the completed Arabidopsis genome using the Arabidopsis arcA protein sequence (At1g18080, NP_173248 [GenBank] ) as a template revealed two homologues, At1g48630 (NP_175296 [GenBank] ) and At3g18130 (NP_188441 [GenBank] ). These three proteins are highly similar to each other (Fig. 1B). RACK1A shares approximately 87% identity and 94% similarity with encoded proteins at loci At1g48630 and At3g18130 and each shares 92% identity and 97% similarity to each other. These three Arabidopsis proteins are approximately 65% identical and 78% similar to mammalian RACK1. The number and position of WD40 repeats, and the PKC binding sites of mammalian RACK1 are also conserved in these Arabidopsis proteins (Fig. 1B). Therefore, hereafter, these three Arabidopsis proteins are designated RACK1A (At1g18080), RACK1B (At1g48630), and RACK1C (At3g18130), respectively. It should be noted that RACK1A was described as a single copy gene in Arabidopsis based on Southern blot analysis (Vahlkamp and Palme, 1997). These three genes also have very similar gene structure, consisting of two exons and one intron (Fig. 1C). Results of gel-based semi-quantitative RT-PCR indicated these three genes have different transcript levels in 10-d-old, light-grown seedlings, with RACK1A mRNA being the most predominant member (Fig. 1D).
RACK1 was originally identified in mammals as an anchoring protein for protein kinase C about a decade ago (Ron et al., 1994), but has now been recognized as a scaffold protein that is able to interact simultaneously with many signalling molecules to regulate diverse signal transduction pathways (see recent review by McCahill et al., 2002). RACK1 is highly conserved in a diverse range of species; besides mammals and plants, RACK1 orthologues were found in our analyses of fish, insects, and fungi EST databases and genomes (see supplementary Fig. 1 at JXB online). The phylogenetic analysis did not reveal a striking difference between Arabidopsis RACK1 and RACK1 proteins from other species (see supplementary Fig. 2 at JXB online). However, most plant RACK1 proteins are distinguishable from RACK1 proteins of non-plant species by an insert of 10-amino acid in the C-terminal portion of the protein (Fig. 1B; see supplementary Fig. 1 at JXB online). Moreover, Arabidopsis RACK1 proteins are also distinguishable from each other within this insert (Fig. 1B).
Within the current NCBI and Phytome databases, RACK1 homologues are present in other plant species such as rice, rape, soybean, tobacco, tomato, beech and alfalfa, as well as algae (see supplementary Fig. 3 at JXB online). Both tobacco and tomato have at least two RACK1 orthologues. Within plants, rape RACK1 (NCBI accession number Q39336) is mostly closed to RACK1A based on phylogenetic analysis (see supplementary Fig. 4 at JXB online).
RACK1 expression
To visualize tissue and organ distribution of RACK1 expression, genomic DNA 2740 bp upstream of the RACK1A start codon, 2215 bp upstream of the RACK1B start codon, and 1091 bp upstream of the RACK1C start codon were each fused with a ß-glucuronidase (GUS) reporter gene and transformed into Arabidopsis (Col-0) plants. RACK1A:GUS, RACK1B:GUS, and RACK1C:GUS displayed very similar expression patterns, and were expressed ubiquitously (Fig. 4A–C). The strongest GUS staining was detected in the meristems of the shoot, primary roots and lateral roots. In reproductive organs, GUS staining was detected in anthers, stigma, and the distal and proximal ends of siliques (Fig. 4A–C). The pattern of RACK1A expression in Arabidopsis based on the RACK1A:GUS transcriptional fusion reporter is largely consistent with that of arcA in tobacco plants analysed by northern analysis with one exception; arcA was detected at a relatively low level in the shoot tips of fully grown tobacco plants (Ishida et al., 1993).
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The ubiquitous expression patterns of the RACK1:GUS transcriptional fusion reporter are also consistent with the microarray data from the Genevestigator Arabidopsis thaliana microarray database (http://www.genevestigator.ethz.ch/; Zimmermann et al., 2004). As shown in Fig. 4 (and in supplementary Fig. 5 at JXB online), RACK1A, RACK1B, and RACK1C transcripts showed very similar expression patterns in the various tissues and organs. However, based on the microarray data, the levels of the transcripts were distinguishable among RACK1 genes, with RACK1A having highest transcript levels in this gene family (see supplementary Fig. 5 at JXB online). Such differences in transcript levels were also observed in 10-d-old, light-grown whole seedlings (Fig. 1D).
rack1a mutants
It has been shown by molecular modelling (Fig. 2) that the Arabidopsis arcA, RACK1A, is unlikely to function as a heterotrimeric G-protein ß subunit and fails to interact with the G subunit in the yeast split-ubiquitin assay (Fig. 3), and the results from sequence and homologue research indicated that Arabidopsis arcA genes are mammalian RACK1 orthologues (Fig. 1). It is interesting to note that RACK1 is a single copy gene in other species whereas some plants have more than two RACK1 orthologues (see supplementary Figs 3 and 4 at JXB online). The focus here was on the functional analysis of RACK1A, the most abundantly-expressed member of the RACK1 gene family in Arabidopsis. T-DNA insertion-mutant alleles of RACK1A were isolated and characterized. rack1a-1, which harbours a T-DNA insertion in the first exon of the RACK1A gene, was obtained by screening deconvoluted pools of DNA from T-DNA transformed Arabidopsis (Col-0) plants as described in the Materials and methods. A second allele, rack1a-2, was obtained from the Salk Institute sequence-indexed insertion mutant collection (http://signal.salk.edu/cgi-bin/tdnaexpress), and the T-DNA insertion was confirmed to be in the first intron of the RACK1A gene (Fig. 5A). RT-PCR analysis using primers specific for RACK1A 5'- and 3'-UTR regions did not detect RACK1 transcript in either allele (Fig. 5B), indicating that the T-DNA insertions probably result in loss-of-function alleles. This notion is supported by the facts that the T-DNA insertion in rack1a-1 tagged a site 41bp downstream of the start codon, presumably from which no functional polypeptide or protein would be encoded, and that rack1a-2 displayed identical phenotypes with rack1a-1 mutant (Figs 5C, D, 6, 7).
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rack1a mutants displayed pleiotropic phenotypes. In etiolated seedlings, the hypocotyls of rack1a mutants were shorter than those of the wild type (Fig. 5C). The short hypocotyl phenotype of rack1a mutants are similar to that of null mutants of Arabidopsis G-protein
subunit, gpa1, and ß subunit, agb1, as reported previously (Ullah et al., 2001, 2003; Jones et al., 2003). However rack1a mutants had closed hooks whereas G-protein mutants had partially-opened hooks. In light-grown seedlings, the cotyledons of rack1a mutant seedlings were epinastic (Fig. 4D, E), resembling the phenotypes of some auxin mutants, such as yucca mutants (Zhao et al., 2001).
rack1a mutants have reduced rate of rosette leaf production
Mature rack1a mutant plants had a smaller rosette size than wild-type plants (Fig. 6C). Based on the number of days taken for bolting, rack1a mutants were late flowering, especially under short-day conditions (Fig. 6A, B). However, rack1a mutants are not classic flowering mutants, based on the well-established criteria for determining a flowering mutant (Weigel and Glazebrook, 2002). rack1a mutants are late flowering mutants in short-day conditions, because it took more than 120 d for rack1a mutants to bolt under short-day conditions, compared with approximately 80 d for wild-type plants. However, rack1a mutants produced about 27 rosette leaves in short-day conditions when bolting, compared with 41 rosette leaves for wild-type plants.
It was found further that rack1a mutants had defects in rosette leaf production. In the vegetative growth stage in prebolting plants, rack1a mutants produced approximately half the number of rosette leaves of wild-type plants (Fig. 6D), resulting in a decrease of the leaf production rate by about 50% (Fig. 6E). These results indicate that leaf development in rack1a mutants is delayed. rack1a mutant leaves had near wild-type morphology although rosette leaves appeared to be slightly narrow and epinastic (Fig. 6D).
rack1a mutants have altered sensitivities to hormones
rack1a mutants displayed altered sensitivities to several plant hormones. The previously established germination assay (Ullah et al., 2002; Chen et al., 2004) was used to test the sensitivities of rack1a mutants to gibberellin (GA) and brassinosteroid. Wild-type and rack1a mutant seeds were pretreated with the GA biosynthesis inhibitor, paclobutrazol (PAC), to reduce endogenous GA, then sown on plates supplemented with defined concentrations of gibberellic acid (GA3) or brassinolide (BL). As shown in Fig. 7, rack1a mutant seeds were less responsive to exogenous GA3 and BL.
The assay of ABA-induced inhibition of seed germination and early seedling development was used to measure the responsiveness of rack1a mutants to ABA. It was found that the ABA responsiveness was enhanced in rack1a mutants in both seed germination and early seedling development (Fig. 7C, D). It was found that when exogenous ABA concentration was lower than 0.5 µM, both wild-type and rack1a mutant seeds could germinate and develop into seedlings, and no significant difference was observed. When ABA was applied at concentrations higher than 3.0 µM, germination and early seedling development was equally inhibited for both wild-type and rack1a mutants. Therefore, it appeared that such a difference in ABA responsiveness between wild-type and rack1a mutants occurred in a narrow range of ABA concentrations. It was also found that rack1a mutants were more sensitive to ABA in the arrest of early seedling development than in the inhibition of seed germination (Fig. 7C, D).
Because arcA, a tobacco RACK1 homologue, was originally identified as an auxin-induced gene, auxin sensitivities of rack1a mutants were examined further. Previously-established lateral and adventitious root formation assays (Ullah et al., 2003) were used to measure the responsiveness of rack1a mutants to auxin. Wild-type and rack1a mutant seedling were pretreated with the auxin transport inhibitor, naphthylphthalamic acid (NPA), then moved to medium containing 0.1 µM 1-NAA. As shown in Fig. 7E, rack1a mutants responded to exogenous 1-NAA, but produced fewer lateral roots compared with wild type. Excised hypocotyls were used for the adventitious root formation assay. The advantage of this assay is that there are no pre-existing root primordia in hypocotyls. It was found that, in response to exogenous 1-NAA, the excised hypocotyls of rack1a mutants produced about 40% fewer adventitious roots than the wild type (Fig. 7F). These results indicate that rack1a mutants are less sensitive to auxin.
In summary, rack1a mutants are less sensitive to GA and BR in seed germination, more responsive to ABA in seed germination and early seedling development, and less responsive to auxin in root formation. Based on the results of cytokinin-mediated root elongation inhibition, and ethylene-induced triple responses, rack1a mutants had near wild-type responsiveness to cytokinin and ethylene (data not shown).
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Discussion |
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Mammalian RACK1 serves as a scaffold protein for a wide range of signalling molecules including kinases and membrane-bound receptors. RACK1 is a WD-repeat family protein and is predicted to have a ß-propeller architecture with seven blades, similar to the heterotrimeric G-protein ß subunit. RACK1 is evolutionally conserved in a wide range of species. In plants, the first RACK1 orthologue was cloned over a decade ago (Ishida et al., 1993). Recently, Chang et al. (2005) found that Arabidopsis orthologues of RACK1 associate with the 40S subunit of cytosolic ribosomes. In another independent study, Giavalisco et al. (2005) found that Arabidopsis RACK1 co-migrated with the 80S ribosomes suggesting a possible association with the 80S ribosomes. These studies providing the first biochemical evidence that the function of RACK1 is conserved in plants, because in yeasts and mammals, it has been shown that RACK1 associates with ribosomes (Link et al., 1999; Shor et al., 2003; Ceci et al., 2003; Nilsson et al., 2004; Sengupta et al., 2004). In the present report, the first direct genetic evidence that Arabidopsis RACK1 has a role in hormonal responses and affect multiple developmental processes is provided.
It was found that loss-of-function of one member of the Arabidopsis RACK1 gene family, RACK1A, conferred developmental defects in both seedlings and mature plants, including hypocotyl and cotyledon growth, and rosette leaf production. rack1a mutants displayed reduced sensitivities to GA and BR in germination response, hypersensitivity to ABA in seed germination and early seedling development, and reduced sensitivity to auxin in adventitious and lateral root formation.
The involvement of RACK1A in multiple hormonal signalling is consistent with a scaffold function, and indicates that RACK1A may interact with a key signalling component in these signalling pathways. For example, rack1a mutants share several phenotypes with Arabidopsis heterotrimeric G-protein mutants, gpa1 and agb1, such as reduced sensitivity to GA and BR in germination, increased sensitivity to ABA in germination, and reduced sensitivity to auxin in root formation (Ullah et al., 2002, 2003). It is possible that RACK1 and the heterotrimeric G-proteins may constitute a signalling complex to regulate these diverse responses. In mammals, RACK1 can physically interact with the Gß dimer (Dell et al., 2002; Chen S et al., 2004a, b), and regulate some specific processes mediated by the Gß dimer. Recently, it has been found that RACK1 protein levels are down-regulated in a heterotrimeric G-protein subunit mutant in rice (Komatsu et al., 2005), implying that plant RACK1 may be indeed regulated by G-proteins, or vice versa. However, RACK1A may have roles in other developmental processes independent of the heterotrimeric G-protein complex. For example, rack1a mutants have reduced rosette leaf production whereas the gpa1 and agb1 mutants appeared to be wild type (data not shown). It is also known that mammalian RACK1 binds to the Gß dimer and has regulatory roles in some processes, such as the inhibition Gß-mediated activation of phospholipase C ß2 and adenylyl cyclase II, but has no effect on many other functions of Gß (Chen S et al., 2004a).
Alternatively, because rack1a mutants have a pleiotropic phenotype and RACK1 has been found to be associated with the 40S subunit of ribosomes in Arabidopsis (Chang et al., 2005), it is possible that RACK1A may have a general role in translational regulation.
A dual role of RACK1 in signalling and translational regulation has already been recognized in mammals and yeasts. RACK1 has been found to be a potential physical and functional linker between various signalling molecules (Nilsson et al., 2004). So far, RACK1 and its orthologues have been found to be associated with the 40S small ribosomal subunit in human (Link et al., 1999), yeasts (Inada et al., 2002; Shor et al., 2003), Arabidopsis (Chang et al., 2005), and algae (Manuell et al., 2005). A recent cryo-electron microscopy (Cryo-EM) study of the 80S ribosome located RACK1 on the head region of 40S subunit, in the immediate vicinity of the mRNA exit site of the ribosome (Sengupta et al., 2004). One of the signalling molecules that interact with RACK1 on the ribosome is the activated PKC (Ceci et al., 2003). Through its interaction with RACK1, activated PKC is able to phosphorylate a translation initiation factor eIF6 and dissociate the latter from free 60S ribosomal subunit. This release of eIF6 from the 60S ribosomal subunit allows the joining of 40S and 60S subunits to form the functional 80S ribosome and hence activate the translation. Another signalling protein Src kinase was also found to bind to ribosome-bound RACK1 (Sengupta et al., 2004). Furthermore, RACK1 was found to be present both in a ribosome-bound and ribosome-non-bound forms in humans (Ceci et al., 2003), and yeasts (Shor et al., 2003; Baum et al., 2004). It is not clear how many of the RACK1 interacting partners identified so far bind to ribosome-bound RACK1 and how many of them bind to non-ribosome-bound RACK1.
Despite three conserved RACK1 paralogues in Arabidopsis, disruption of a single gene, RACK1A, from this gene family is sufficient to cause defects in many developmental processes. This indicates that RACK1A may have a distinguishable function from the other two members, or that RACK1A is required for the function of the other two members. Because there are three RACK1 proteins in Arabidopsis, it is possible that these proteins can form heterodimers through the WD40 protein–protein interaction domains. Such interactions between WD40 repeat proteins have already been described. For example, bovine Gß binds at least three WD40 repeat proteins including RACK1 (Dell et al., 2002). Mammalian RACK1 binds to WD40 repeat protein FAN, a factor associated with neutral sphingomyelinase activation (Tcherkasowa et al., 2002). Two TATA-binding protein (TBP)-associated factors (TAF) TAF72 and TAF73, both of which are WD40 repeat proteins, co-immuoprecipitated in yeast (Mitsuzawa et al., 2001). Both RACK1A and RACK1B were found to be associated with the 40S subunit of cytosolic ribosomes in Arabidopsis (Chang et al., 2005). Such a heterodimerization among WD40 repeat proteins could have an important role for integrating signals from different cellular processes (Chen et al., 2004b), and could be required for the RACK1 function in Arabidopsis.
Mammalian RACK1 is known to interact with PKC. While no obvious PKC orthologue, the classic RACK1 interacting partner, is found in the sequenced plant genomes, it is possible that plant RACK1 interacts with a protein kinase that may share some property with PKC, such as regulation by lipids. For example, some members of the large plant AGC protein kinase family (S6 kinase, IRE, NDR) have a PKC-like kinase extension domain (Bogre et al., 2003).
In summary, these genetic data support that RACK1A mediates hormonal responses, and has a regulatory role in multiple developmental processes. RACK1A may either represent a critical integrative point for hormone signalling pathways, or is an essential component for translational regulation for genes that are involved in these hormone responses or developmental processes, or both. At this point, neither possibility could be ruled out. Future studies should focus on the identification of other RACK1 interacting partners and of RACK1 affected genes in plants.
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Acknowledgements |
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This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation, the British Columbia Ministry of Advanced Education, the University of British Columbia, and the National Natural Science Foundation of China (grant no. 30528023) to J-GC; by grants from the NIGMS (GM65989-01) and the NSF (MCB-0209711) to AMJ, and in part by a grant from the National Key Basic Research and Development Program of China (2003CB114300303) to JL. Isolation of the rack1a-1 and rack1a-2 alleles was made possible by an NSF grant (0115103) to JRE.
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Present address: Department of Biology, Howard University, Washington, DC 20059, USA. 
Present address: Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695, USA.
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