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RACK1 genes regulate plant development with unequal genetic redundancy in Arabidopsis.

Guo J, Chen JG - BMC Plant Biol. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Botany, University of British Columbia, Vancouver, BC, Canada. jimguo@interchange.ubc.ca

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

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The expression of RACK1 genes in rack1a, rack1b and rack1c single and double mutants. (A) RT-PCR analysis of the expression of RACK1 genes in rack1a, rack1b and rack1c single and double mutants. RT-PCR was performed at 28 cycles. The expression of ACTIN2 was used as a control. (B) Quantitative real-time PCR analysis of the transcript level of RACK1 genes in rack1a, rack1b and rack1c single and double mutants. The transcript level of RACK1 genes was normalized against the transcript level of ACTIN2 in each sample. The relative transcript level of RACK1 genes in mutant backgrounds was compared with that in wild-type (Col) (set as 1). Shown are the averages of three replicates ± S.D.
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Figure 7: The expression of RACK1 genes in rack1a, rack1b and rack1c single and double mutants. (A) RT-PCR analysis of the expression of RACK1 genes in rack1a, rack1b and rack1c single and double mutants. RT-PCR was performed at 28 cycles. The expression of ACTIN2 was used as a control. (B) Quantitative real-time PCR analysis of the transcript level of RACK1 genes in rack1a, rack1b and rack1c single and double mutants. The transcript level of RACK1 genes was normalized against the transcript level of ACTIN2 in each sample. The relative transcript level of RACK1 genes in mutant backgrounds was compared with that in wild-type (Col) (set as 1). Shown are the averages of three replicates ± S.D.

Mentions: The analysis of the expression patterns and transcript level of three RACK1 genes in various tissues and organs supported the view that the unequal genetic redundancy of RACK1 genes is likely due to the difference in the gene expression level. However, other possibilities may also exist. For example, as reviewed by Briggs et al. (2006), cross-regulation is another mechanism that attributes to the unequal genetic redundancy of some homologous genes [16]. Because RACK1A, RACK1B and RACK1C are approximately 90% identical to each other at the amino acid level, we were unable to obtain antibodies that can specifically recognize each RACK1 protein. Therefore, in this study, we examined the impact of loss-of-function mutations of each RACK1 gene on the transcription of the other two RACK1 genes. Further, we examined the impact of combination of loss-of-function mutations of two RACK1 genes on the transcription of the other RACK1 gene. Specifically, we examined the transcript level of RACK1A in rack1b and rack1c single and double mutants, the transcript level of RACK1B in rack1a and rack1c single and double mutants, and the transcript level of RACK1C in rack1a and rack1b single and double mutants, and compared with their transcript levels in wild-type. For this analysis, we used the 4.5 d-old, light-grown whole seedlings. By using RT-PCR, we noticed that the transcript level of RACK1B was reduced in rack1a and rack1c single and double mutants (Figure 7A). Similarly, the transcript level of RACK1C was reduced in rack1a and rack1b single and double mutants (Figure 7A). However, we did not observe a dramatic reduction of the transcript level of RACK1A in rack1b and rack1c single and double mutants, compared with that in wild-type (Figure 7A). Because the transcript level of RACK1A is the most abundant among three RACK1 homologous genes and the conditions used for RT-PCR (e.g. PCR at 28 cycles) may not allow us to visualize any differences in RACK1A transcript level among different samples, subsequently we used quantitative real-time PCR to more accurately compare the transcript level of three RACK1 genes in wild-type and mutants. We found that the transcript level of any given RACK1 gene was reduced in the loss-of-function alleles of each and both of the other two RACK1 genes (Figure 7B).


RACK1 genes regulate plant development with unequal genetic redundancy in Arabidopsis.

Guo J, Chen JG - BMC Plant Biol. (2008)

The expression of RACK1 genes in rack1a, rack1b and rack1c single and double mutants. (A) RT-PCR analysis of the expression of RACK1 genes in rack1a, rack1b and rack1c single and double mutants. RT-PCR was performed at 28 cycles. The expression of ACTIN2 was used as a control. (B) Quantitative real-time PCR analysis of the transcript level of RACK1 genes in rack1a, rack1b and rack1c single and double mutants. The transcript level of RACK1 genes was normalized against the transcript level of ACTIN2 in each sample. The relative transcript level of RACK1 genes in mutant backgrounds was compared with that in wild-type (Col) (set as 1). Shown are the averages of three replicates ± S.D.
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Figure 7: The expression of RACK1 genes in rack1a, rack1b and rack1c single and double mutants. (A) RT-PCR analysis of the expression of RACK1 genes in rack1a, rack1b and rack1c single and double mutants. RT-PCR was performed at 28 cycles. The expression of ACTIN2 was used as a control. (B) Quantitative real-time PCR analysis of the transcript level of RACK1 genes in rack1a, rack1b and rack1c single and double mutants. The transcript level of RACK1 genes was normalized against the transcript level of ACTIN2 in each sample. The relative transcript level of RACK1 genes in mutant backgrounds was compared with that in wild-type (Col) (set as 1). Shown are the averages of three replicates ± S.D.
Mentions: The analysis of the expression patterns and transcript level of three RACK1 genes in various tissues and organs supported the view that the unequal genetic redundancy of RACK1 genes is likely due to the difference in the gene expression level. However, other possibilities may also exist. For example, as reviewed by Briggs et al. (2006), cross-regulation is another mechanism that attributes to the unequal genetic redundancy of some homologous genes [16]. Because RACK1A, RACK1B and RACK1C are approximately 90% identical to each other at the amino acid level, we were unable to obtain antibodies that can specifically recognize each RACK1 protein. Therefore, in this study, we examined the impact of loss-of-function mutations of each RACK1 gene on the transcription of the other two RACK1 genes. Further, we examined the impact of combination of loss-of-function mutations of two RACK1 genes on the transcription of the other RACK1 gene. Specifically, we examined the transcript level of RACK1A in rack1b and rack1c single and double mutants, the transcript level of RACK1B in rack1a and rack1c single and double mutants, and the transcript level of RACK1C in rack1a and rack1b single and double mutants, and compared with their transcript levels in wild-type. For this analysis, we used the 4.5 d-old, light-grown whole seedlings. By using RT-PCR, we noticed that the transcript level of RACK1B was reduced in rack1a and rack1c single and double mutants (Figure 7A). Similarly, the transcript level of RACK1C was reduced in rack1a and rack1b single and double mutants (Figure 7A). However, we did not observe a dramatic reduction of the transcript level of RACK1A in rack1b and rack1c single and double mutants, compared with that in wild-type (Figure 7A). Because the transcript level of RACK1A is the most abundant among three RACK1 homologous genes and the conditions used for RT-PCR (e.g. PCR at 28 cycles) may not allow us to visualize any differences in RACK1A transcript level among different samples, subsequently we used quantitative real-time PCR to more accurately compare the transcript level of three RACK1 genes in wild-type and mutants. We found that the transcript level of any given RACK1 gene was reduced in the loss-of-function alleles of each and both of the other two RACK1 genes (Figure 7B).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Botany, University of British Columbia, Vancouver, BC, Canada. jimguo@interchange.ubc.ca

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus