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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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T-DNA insertional mutants of RACK1B and RACK1C. (A) A diagram to illustrate the T-DNA insertion sites in rack1b-1 and rack1b-2 mutants. (B) RT-PCR analysis of RACK1B transcript in rack1b mutants. RACK1B-specific primers that amplify the full-length transcript of RACK1B in wild-type (Col) were used. (C) The rosette morphology of rack1b and rack1c mutants. Shown are plants grown 48 days under 10/14 h photoperiod. (D) A diagram to illustrate the T-DNA insertion sites in rack1c-1 and rack1c-2 mutants. (E) RT-PCR analysis of RACK1C transcript in rack1c mutants.RACK1C-specific primers that amplify the full-length transcript of RACK1C in Col were used. Gray boxes in (A) and (D) represent coding regions and white boxes represent 5'-UTR and 3'-UTR regions. The T-DNA inserts are not drawn to scale. LB, T-DNA left border. Total RNA isolated from 10 d-old, light-grown seedlings was used for RT-PCR analysis in (B) and (E). RT-PCR was performed with 30 cycles. The expression of ACTIN2 was used as a control.
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Figure 2: T-DNA insertional mutants of RACK1B and RACK1C. (A) A diagram to illustrate the T-DNA insertion sites in rack1b-1 and rack1b-2 mutants. (B) RT-PCR analysis of RACK1B transcript in rack1b mutants. RACK1B-specific primers that amplify the full-length transcript of RACK1B in wild-type (Col) were used. (C) The rosette morphology of rack1b and rack1c mutants. Shown are plants grown 48 days under 10/14 h photoperiod. (D) A diagram to illustrate the T-DNA insertion sites in rack1c-1 and rack1c-2 mutants. (E) RT-PCR analysis of RACK1C transcript in rack1c mutants.RACK1C-specific primers that amplify the full-length transcript of RACK1C in Col were used. Gray boxes in (A) and (D) represent coding regions and white boxes represent 5'-UTR and 3'-UTR regions. The T-DNA inserts are not drawn to scale. LB, T-DNA left border. Total RNA isolated from 10 d-old, light-grown seedlings was used for RT-PCR analysis in (B) and (E). RT-PCR was performed with 30 cycles. The expression of ACTIN2 was used as a control.

Mentions: Arabidopsis genome contains three RACK1 homologous genes, designated as RACK1A, RACK1B and RACK1C, respectively [15]. Within the RACK1 gene family, mutant alleles for only RACK1A have been reported previously [15]. We report here the isolation and characterization of rack1b and rack1c mutant alleles. By searching the Salk Institute sequence-indexed insertion mutant collection , we obtained two independent T-DNA insertional alleles for each RACK1 gene. All alleles are in the Columbia (Col-0) ecotypic background. We designated the two mutant alleles for RACK1B as rack1b-1 and rack1b-2, respectively. In rack1b-1 allele, the T-DNA was inserted in the second exon of RACK1B gene, and in the rack1b-2 allele, the T-DNA was inserted in the first intron (Figure 2A). RT-PCR analysis indicated that the full-length transcript of RACK1B was absent in both alleles (Figure 2B), implying that they are likely loss-of-function alleles. Unlike rack1a mutants, rack1b mutants do not display any apparent developmental defects (Figure 2C). We designated the two mutant alleles for RACK1C as rack1c-1 and rack1c-2, respectively (Figure 2D). In rack1c-1 allele, the T-DNA was inserted in the second exon of RACK1C gene, and in the rack1c-2 allele, the T-DNA was inserted in the 5'-UTR region. RT-PCR analysis indicated that the full-length transcript of RACK1C was absent in both alleles (Figure 2E), implying that they are likely loss-of-function alleles. Similar to rack1b mutants but unlike rack1a mutants, rack1c mutants do not display any apparent defects in plant development (Figure 2C).


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

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

T-DNA insertional mutants of RACK1B and RACK1C. (A) A diagram to illustrate the T-DNA insertion sites in rack1b-1 and rack1b-2 mutants. (B) RT-PCR analysis of RACK1B transcript in rack1b mutants. RACK1B-specific primers that amplify the full-length transcript of RACK1B in wild-type (Col) were used. (C) The rosette morphology of rack1b and rack1c mutants. Shown are plants grown 48 days under 10/14 h photoperiod. (D) A diagram to illustrate the T-DNA insertion sites in rack1c-1 and rack1c-2 mutants. (E) RT-PCR analysis of RACK1C transcript in rack1c mutants.RACK1C-specific primers that amplify the full-length transcript of RACK1C in Col were used. Gray boxes in (A) and (D) represent coding regions and white boxes represent 5'-UTR and 3'-UTR regions. The T-DNA inserts are not drawn to scale. LB, T-DNA left border. Total RNA isolated from 10 d-old, light-grown seedlings was used for RT-PCR analysis in (B) and (E). RT-PCR was performed with 30 cycles. The expression of ACTIN2 was used as a control.
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Figure 2: T-DNA insertional mutants of RACK1B and RACK1C. (A) A diagram to illustrate the T-DNA insertion sites in rack1b-1 and rack1b-2 mutants. (B) RT-PCR analysis of RACK1B transcript in rack1b mutants. RACK1B-specific primers that amplify the full-length transcript of RACK1B in wild-type (Col) were used. (C) The rosette morphology of rack1b and rack1c mutants. Shown are plants grown 48 days under 10/14 h photoperiod. (D) A diagram to illustrate the T-DNA insertion sites in rack1c-1 and rack1c-2 mutants. (E) RT-PCR analysis of RACK1C transcript in rack1c mutants.RACK1C-specific primers that amplify the full-length transcript of RACK1C in Col were used. Gray boxes in (A) and (D) represent coding regions and white boxes represent 5'-UTR and 3'-UTR regions. The T-DNA inserts are not drawn to scale. LB, T-DNA left border. Total RNA isolated from 10 d-old, light-grown seedlings was used for RT-PCR analysis in (B) and (E). RT-PCR was performed with 30 cycles. The expression of ACTIN2 was used as a control.
Mentions: Arabidopsis genome contains three RACK1 homologous genes, designated as RACK1A, RACK1B and RACK1C, respectively [15]. Within the RACK1 gene family, mutant alleles for only RACK1A have been reported previously [15]. We report here the isolation and characterization of rack1b and rack1c mutant alleles. By searching the Salk Institute sequence-indexed insertion mutant collection , we obtained two independent T-DNA insertional alleles for each RACK1 gene. All alleles are in the Columbia (Col-0) ecotypic background. We designated the two mutant alleles for RACK1B as rack1b-1 and rack1b-2, respectively. In rack1b-1 allele, the T-DNA was inserted in the second exon of RACK1B gene, and in the rack1b-2 allele, the T-DNA was inserted in the first intron (Figure 2A). RT-PCR analysis indicated that the full-length transcript of RACK1B was absent in both alleles (Figure 2B), implying that they are likely loss-of-function alleles. Unlike rack1a mutants, rack1b mutants do not display any apparent developmental defects (Figure 2C). We designated the two mutant alleles for RACK1C as rack1c-1 and rack1c-2, respectively (Figure 2D). In rack1c-1 allele, the T-DNA was inserted in the second exon of RACK1C gene, and in the rack1c-2 allele, the T-DNA was inserted in the 5'-UTR region. RT-PCR analysis indicated that the full-length transcript of RACK1C was absent in both alleles (Figure 2E), implying that they are likely loss-of-function alleles. Similar to rack1b mutants but unlike rack1a mutants, rack1c mutants do not display any apparent defects in plant development (Figure 2C).

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