RACK1 genes regulate plant development with unequal genetic redundancy in Arabidopsis
© Guo and Chen; licensee BioMed Central Ltd. 2008
Received: 12 August 2008
Accepted: 23 October 2008
Published: 23 October 2008
RACK1 is a versatile scaffold protein in mammals, regulating diverse developmental processes. Unlike in non-plant organisms where RACK1 is encoded by a single gene, Arabidopsis genome contains three RACK1 homologous genes, designated as RACK1A, RACK1B and RACK1C, respectively. Previous studies indicated that the loss-of-function alleles of RACK1A displayed multiple defects in plant development. However, the functions of RACK1B and RACK1C remain elusive. Further, the relationships between three RACK1 homologous genes are unknown.
We isolated mutant alleles with loss-of-function mutations in RACK1B and RACK1C, and examined the impact of these mutations on plant development. We found that unlike in RACK1A, loss-of-function mutations in RACK1B or RACK1C do not confer apparent defects in plant development, including rosette leaf production and root development. Analyses of rack1a, rack1b and rack1c double and triple mutants, however, revealed that rack1b and rack1c can enhance the rack1a mutant's developmental defects, and an extreme developmental defect and lethality were observed in rack1a rack1b rack1c triple mutant. Complementation studies indicated that RACK1B and RACK1C are in principle functionally equivalent to RACK1A. Gene expression studies indicated that three RACK1 genes display similar expression patterns but are expressed at different levels. Further, RACK1 genes positively regulate each other's expression.
These results suggested that RACK1 genes are critical regulators of plant development and that RACK1 genes function in an unequally redundant manner. Both the difference in RACK1 gene expression level and the cross-regulation are likely the molecular determinants of their unequal genetic redundancy.
Receptor for activated C kinase 1 (RACK1) is a seven tryptophan-aspartic acid-domain (WD40) repeat-containing protein, and was originally identified as an anchoring protein for protein kinase C (PKC) in mammals, shuttling the activated enzyme to different subcellular sites [1, 2]. Structurally, RACK1 is similar to the heterotrimeric G-protein β subunit (Gβ) which has a seven-bladed propeller structure with one WD40 unit constituting each blade (reviewed in [3, 4]). Increasing evidence suggests that in addition to binding the activated PKC, mammalian RACK1 functions as a scaffold protein by physically interacting with many other proteins and facilitating their interactions. It has been shown that RACK1 plays regulatory roles in diverse developmental and physiological responses, including cell cycle control, cell movement and growth, immune response, and neural responses in mammals (reviewed in [3, 4]). Therefore, RACK1 is now viewed as a versatile scaffold protein, serving as a nexus for multiple signal transduction pathways.
Although not recognized as such, the first plant RACK1 gene was cloned from tobacco BY-2 cells as an auxin (2,4-dichlorophenoxyacetic acid, 2,4-D) inducible gene, arcA . Subsequently, the amino acid sequence homologues of RACK1 were found in all plant species examined (reviewed in ). Earlier studies based on gene expression and induction analysis implied that plant RACK1 may have a role in hormone-mediated cell division [5, 7], UV and salicylic acid responses . In rice, RACK1, named RWD , was found to be one of the seven proteins whose expressions were down-regulated in d1 mutant, a loss-of-function allele of rice heterotrimeric G-protein α subunit . Further, rice RACK1 protein was induced by abscisic acid (ABA) in imbibed wild-type seeds, but not in d1 mutant seeds. It was proposed that RACK1 may play a role in rice embryogenesis and germination . Furthermore, recently, it has been demonstrated that RACK1 proteins are key regulators of innate immunity by interacting with multiple proteins in the Rac1 immune complex in rice . In Arabidopsis, RACK1 proteins have been found to be associated with the subunits of ribosomes [12, 13], but no signaling proteins have been identified to interact with Arabidopsis RACK1 proteins.
T-DNA insertional mutants of RACK1B and RACK1C
Loss-of-function mutations in RACK1B and RACK1Cenhance the developmental defects in rosette leaf production of rack1a mutant
Subsequently, we generated rack1a-1 rack1b-2 rack1c-1 triple mutant. Very few triple mutants could survive in soil. For those survived, they were extremely slow in growth and development, and produced fewest rosette leaves and smallest rosette size among all genotypes examined (Figure 3A–D). Not surprisingly, the rate of rosette leaf production in the triple mutant was the slowest among all genotypes examined (Figure 3C). Because rack1a-1 rack1b-2 rack1c-1 triple mutants could not survive to maturity to produce seeds, these triple mutants were maintained in plants homozygous for the rack1b-2 and rack1c-1 loci and heterozygous for the rack1a-1 locus. Because rack1b-2 rack1c-1 double mutants had wild-type morphology whereas rack1a-1 rack1b-2 rack1c-1 had extreme pleiotropic phenotype, rack1a-1 rack1b-2 rack1c-1 triple mutants can be readily picked up from the segregating progeny of plants homozygous for the rack1b-2 and rack1c-1 loci and heterozygous for the rack1a-1 locus.
Loss-of-function mutations in RACK1B and RACK1Cenhance the defects in root development of rack1a mutant
Genetic complementation of rack1a mutants by overexpressing RACK1genes
Expression of Arabidopsis RACK1genes
In order to quantify the difference in transcript level of RACK1A, RACK1B and RACK1C genes, we used quantitative real-time PCR to more accurately compare the transcript level of three RACK1 genes in different tissues and organs of wild-type Col plants. We selected the samples of shoots and roots of 4 d- and 7 d-old light-grown seedlings and rosette leaves and roots of mature plants for quantitative real-time PCR analysis. We found that consistent with the result of RT-PCR analysis, the transcript level of RACK1C was the lowest and that of RACK1A was the highest among three RACK1 genes, with a trend of RACK1A > RACK1B > RACK1C in all samples examined (Figure 6B). For example, the transcript level of RACK1A was about 5-fold higher than that of RACK1C in the roots of 4 d-old, light-grown seedlings (Figure 6B). In this sample, the transcript level of RACK1B was approximately 2-fold higher than that of RACK1C.
Cross-regulation of RACK1genes at the transcription level
Roles of RACK1genes in plant development
RACK1 gene is evolutionarily conserved in diverse organisms. Although the research interest in RACK1 has grown exponentially since its discovery  and RACK1 is now viewed as a multi-functional, versatile scaffold protein in mammals and in yeasts (reviewed in [3, 4]), the function of RACK1 in plants remains poorly understood. We are just starting to have some hints about its potential functions in plants. Preliminary analysis suggested RACK1 may mediate multiple hormone responses and developmental processes in Arabidopsis . In this study, we focused on the two characteristic developmental defects of rack1a mutants, namely the reduction in rosette leaf production and the reduction in primary root growth and lateral root formation, to study the function of RACK1B and RACK1C and the genetic relationship between RACK1 homologous genes in plant development. We demonstrated that RACK1 genes are critical regulators of plant development and are essential for plant survival. Simultaneous disruption of the function of all three RACK1 genes results in lethality. Thanks to the unequal genetic redundancy of RACK1 genes, we are still able to study the role of RACK1 genes in plant development. The rack1a single mutants, rack1a rack1b and rack1a rack1c double mutants all display developmental defects and are viable. Therefore, these mutants can be treated as "weak alleles" of rack1 mutants. Now that we have identified RACK1 genes as critical regulators of plant development and all "weak alleles" of rack1 mutants are available, future studies should focus on the elucidation of the molecular mechanism by which RACK1 genes regulate plant development, including rosette leaf production, root growth and lateral root formation. Because rack1a mutants have also been shown to display altered responses to hormones , it remains unclear if the developmental defects observed in rack1 mutants are due to the altered responses to multiple hormones and if there is also unequal genetic redundancy of RACK1 genes in mediating hormone responses. This is a fertile area that is worth further investigation.
Mechanism of unequal genetic redundancy of RACK1genes
Genetic redundancy of homologous genes is thought to be due to gene duplication events during the evolution of the organism. Between homologous genes, genetic redundancy can be classified as full redundancy, partial redundancy, and unequal redundancy . While full redundancy and partial redundancy have been documented in numerous cases, unequal genetic redundancy has just begun to be recognized as a common phenomenon of genetic relationship of homologous genes . Unlike non-plant organisms whose genomes contain only a single RACK1 gene, some plant genomes contain more than one RACK1 genes (Figure 1). In particular, the Arabidopsis genome contains three RACK1 genes, which share the similar gene structure with two exons and one intron, and encode three highly similar proteins with approximately 90% identity at the amino acid level . However, the relationship between three Arabidopsis RACK1 homologous genes has been unknown. Previously, we showed that loss-of-function mutation in one member of Arabidopsis RACK1A genes, RACK1A, conferred multiple defects in plant development . Here we show that loss-of-function mutations in RACK1B or RACK1C do not confer apparent developmental defects (Figure 2). These results suggested that RACK1B and RACK1C are likely dispensable in plant development. However, we found that although rack1b and rack1c mutants displayed wild-type morphology, rack1b and rack1c can strongly enhance the developmental defects of rack1a mutants (Figure 3, Figure 4). These results suggested that RACK1B and RACK1C still contribute significantly to the overall activity of RACK1 genes. Because the significance of the RACK1B and RACK1C is determined via the mutants, not directly in the wild-type plants, it is also possible that in the wild-type plants, all the function of RACK1 genes is explicated by RACK1A with no contribution from RACK1B or RACK1C and the these latter can play a role only if RACK1A is not present (e.g. in the rack1a mutant). Nonetheless, the behaviors and relationship of rack1 mutants satisfy the key criteria for RACK1 genes being unequally redundant homologous genes .
The unequal genetic redundancy is caused by many factors. Among them, the difference in gene expression pattern, expression level and cross-regulation of homologous genes have been recognized as major determinants . The unequal genetic redundancy of some homologous genes is mainly due to the difference in expression pattern and/or expression level. For example, CAULIFLOWER (CAL) is closely related in sequence to APETALA1 (AP1), but AP1 and CAL regulate the formation of floral meristem in an unequally redundant manner because AP1 is expressed at much higher level than CAL throughout sepal and petal development . The unequal genetic redundancy of homologous genes can also be primarily due to the cross-regulation. For example,LONG HYPOCOTYL 5 (HY5) and its close homolog HY5 HOMOLOG (HYH), both of which are regulators of photomorphogenesis, are a pair of unequally redundant genes with similar expression patterns and levels [16, 18], but a normal protein expression and activity of HYH was dependent on the presence of a functional HY5 .
In order to get insight into the mechanism of unequal genetic redundancy of three RACK1 genes, we examined each of these possibilities. Firstly, we showed that RACK1B and RACK1C are likely in principle functionally equivalent to RACK1A, because overexpression of either RACK1B or RACK1C under the constitutive CaMV 35S promoter fully complemented the developmental defects of rack1a mutants (Figure 5). Ideally, it would be advantageous to use the native RACK1A promoter to assess the extent of functional equivalency. Nonetheless, results from our complementation studies indicated that overexpression of RACK1B or RACK1C can restore rack1a mutant to wild-type equally well as overexpression of RACK1A, supporting the view that RACK1B and RACK1C likely function similarly as RACK1A. These results implied that the unequal genetic redundancy of RACK1 genes is likely due to the difference in gene expression pattern and/or expression level, rather than the difference in protein sequence or activity. To examine this possibility directly, we found that three RACK1 genes are widely expressed in various tissues and organs in young seedlings and in mature plants (Figure 6). However, RACK1 genes are expressed at different levels with a general trend of RACK1A > RACK1B > RACK1C in all tissues and organs examined (Figure 6). These results supported the view that the difference in gene expression level attributes to the unequal genetic redundancy of RACK1 genes in plant development. However, these results cannot rule out the possibility that the expression of each RACK1 gene may also be restricted to certain cell types. For example, BRL1 and BRL3 are homologous to BRI1, a receptor for brassinosteroid (BR), and function as BR receptors in vascular differentiation in Arabidopsis . It was found that BRI1 is ubiquitously expressed in growing cells, but the expression of BRL1 and BRL3 is restricted to non-overlapping subsets of vascular cells. Future expression analysis at cell level (e.g. by in situ hybridization and reporter GFP analyses) may help address the possibility of cell type-specific expression of RACK1 genes.
We also explored the possibility of cross-regulation by examining the transcript level of each RACK1 gene in the loss-of-function alleles of each or both of the other two RACK1 genes. We found that the transcript level of any given RACK1 gene was reduced in the single or double mutants for the other two RACK1 genes (Figure 7). Therefore, both the difference in gene expression level and the cross-regulation contribute to the unequal genetic redundancy of RACK1 genes. Unlike HY5 and HYH, for which the expression of the duplicate gene (HYH) depends on the presence of the ancestral gene (HY5) , RACK1 homologous genes mutually depend on each other for reaching full expression, adding another level of complexity for the unequal genetic redundancy. The molecular basis of such mutual cross-regulation of RACK1 genes is presently unknown. It would be interesting to test if RACK1 proteins can work together in a complex, for instance, through homo- and hetero-dimerization.
Plant materials and growth conditions
All mutants are in the Arabidopsis Columbia (Col-0) ecotype background. The rack1a-1 and rack1a-2 mutants have been reported previously . Plants were grown in 5 × 5 cm pots containing moistened 1 : 3 mixture of Sunshine Mix #1 (Sun Gro Horticulture Canada Ltd., http://www.sungro.com) and Metro-Mix 220 (W.R. Grace & Co., http://www.grace.com) with 10/14 h (short-day conditions) or 14/10 h (long-day conditions) photoperiod at approximately 120 μmol m-2 s-1 at 23°C.
Isolation of rack1b and rack1cT-DNA insertional mutants
The T-DNA insertion mutants of RACK1B (At1g48630), rack1b-1 (SALK_117422) and rack1b-2 (SALK_145920), and the T-DNA insertion mutants of RACK1C (At3g18130), rack1c-1 (SAIL_199_A04) and rack1c-2 (SALK_017913), were identified from the SALK T-DNA Express database http://signal.salk.edu/cgi-bin/tdnaexpress. For the SALK T-DNA insertional mutants , the insertion was confirmed by PCR using RACK1B-specific primers (5'-TCTCGACCTCAAACCCTG-3' and 5'-GAGAAGACTTTAGAGTCGATGGA-3') or RACK1C-specific primers (5'-ATCTCTCGCTCTGTTACGC-3' and 5'-ACAATACTGACGCAGTCTGG-3') and a T-DNA left border-specific primer JMLB1 (5'-GGCAATCAGCTGTTGCCCGTCTCACTGGTG-3'). For the SAIL T-DNA insertion mutants , a different T-DNA left border-specific primer, GarlicLB3 (5'-TAGCATCTGAATTTCATAACCAATCTCGATACAC-3'), was used. The absence of full-length transcript of RACK1B or RACK1C in these alleles was confirmed by RT-PCR.
Generation of rack1a, rack1b and rack1cdouble and triple mutants
Double mutants between rack1a-1 and rack1b-2 or rack1c-1 were generated by crossing rack1b-2 or rack1c-1 into rack1a-1 single mutant and isolated in the F2 progeny by PCR genotyping. Similarly, double mutants between rack1b-2 and rack1c-1 were generated by crossing rack1c-1 into rack1b-2 single mutant and isolated in the F2 progeny by PCR genotyping. For simplicity, the rack1a rack1b, rack1a rack1c and rack1b rack1c double mutant nomenclatures in this report refer specifically to the rack1a-1 rack1b-2, rack1a-1 rack1c-1 and rack1b-2 rack1c-1 mutants, respectively.
Triple mutant among rack1a-1, rack1b-2 and rack1c-1 was generated by crossing rack1b-2 rack1c-1 into rack1a-1 rack1b-2 double mutants. Because rack1a-1 rack1b-2 rack1c-1 triple mutants cannot survive in soil to maturity, they are maintained in plants homozygous for the rack1b-2 and rack1c-1 loci and heterozygous for the rack1a-1 locus. The status of triple mutant was confirmed by PCR genotyping.
The full-length open-reading frames of RACK1A (At1g18080), RACK1B and RACK1C were amplified from a cDNA library made from seedlings grown in light for 10 d, cloned into the pENTR/D-TOPO vector (Invitrogen, http://www.invitrogen.com), and then subcloned into Gateway plant transformation destination binary vector pB2GW7  by LR recombination reactions. In these constructs, the expression of RACK1A, RACK1B or RACK1C was driven by the 35S promoter of the Cauliflower mosaic virus. Binary vectors were transformed into rack1a-1 or rack1a-2 mutants by Agrobacterium-mediated transformation . At least 16 independent transgenic lines were selected from each transformation, and two to four representative lines were used for further studies. The expression of transgene was examined by RT-PCR.
RNA isolation, RT-PCR and quantitative real-time PCR analyses
For tissue/organ expression pattern analysis, total RNA was isolated from different parts of seedlings or mature plants, using the TRIzol reagent (Invitrogen). cDNA was synthesized from 1 μg total RNA by oligo(dT)20-primed reverse transcription, using THERMOSCRIPT RT (Invitrogen). RACK1A-specific primers (5'-GGCATCTCCAGACACCGAAA-3' and 5'-GCAGAGAGCAACGACAGC-3'), RACK1B-specific primers (5'-TCTCGACCTCAAACCCTG-3' and 5'-GAGAAGACTTTAGAGTCGATGGA-3'), and RACK1C-specific primers (5'-ATCTCTCGCTCTGTTACGC-3' and 5'-ACAATACTGACGCAGTCTGG-3') were used to amplify the transcripts of these three genes, respectively. The expression of ACTIN2 (amplified by primers 5'-GTTGGGATGAACCAGAAGGA-3' and 5'-GAACCACCGATCCAGACACT-3') was used as a control in PCR reactions. For the examination of the transcript level of RACK1A, RACK1B and RACK1C in the T-DNA insertional mutants or in the transgenic lines, 10 d-old, light-grown seedlings were used for total RNA isolation.
For the quantitative analysis of RACK1A, RACK1B and RACK1C transcript levels in the different tissues/organs of wild-type Col plants or in the rack1a-1, rack1b-2 and rack1c-1 single and double mutants, real-time PCR was performed. RACK1A-specific real-time PCR primers (5'-CTGAGGCTGAAAAGGCTGACAACAG-3' and 5'-CTAGTAACGACCAATACCCCAAACTC-3'), RACK1B-specific real-time PCR primers (5'-GGTTCTACTGGAATCGGAAACAAGACC-3' and 5'-CTAGTAACGACCAATACCCCAGACCC-3'), and RACK1C-specific real-time PCR primers (5'-GCAGAGAAGAATGAAGGTGGTGT-3' and 5'-CTAGTAACGACCAATACCCCAGACCC-3') were used. The expression of ACTIN2 (amplified by real-time PCR primers 5'-CCAGAAGGATGCATATGTTGGTGA-3'and 5'-GAGGAGCCTCGGTAAGAAGA-3') was used to normalize the expression of each gene. The quantitative real-time PCR was performed using the MJ MiniOpticon real-time PCR system (Bio-Rad, http://www.biorad.com) and IQ SYBR Green Supermix (Bio-Rad).
Rosette leaf production assay
The number of rosette leaves was collected from wild-type Col and mutant plants grown under 10/14 h or 14/10 h photoperiod with approximately 120 μmol m-2 s-1 at 23°C. At least four plants from each genotype were used in each experiment, and the experiment was repeated twice. The rate of rosette leaf production was expressed as the number of rosette leaves divided by the age of plant.
Root growth assay
Seedlings were grown on MS/G plates consisting of 1/2 Murashige & Skoog (MS) basal medium supplemented with vitamins (Plantmedia, http://www.plantmedia.com), 1% (w/v) sucrose and 0.6% (w/v) phytoagar (Plantmedia), with pH adjusted to 5.7 with 1N KOH. The plates were placed under 14/10 h photoperiod with approximately 120 μmol m-2 s-1 at 23°C with a vertical orientation for monitoring root growth. The length of primary and the number of lateral roots were collected from at least 15 seedlings each genotype.
We thank the Salk Institute Genomic Analysis Laboratory (La Jolla, CA), the Syngenta Biotechnology, Inc. (Research Triangle Park, NC), and the Arabidopsis Biological Resources Center (Columbus, Ohio) for providing the rack1b and rack1c T-DNA insertional mutants. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (grant No. RGPIN311651-05) and the Canada Foundation for Innovation (grant No. 10496).
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