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The Tinkerbell (Tink) Mutation Identifies the Dual-Specificity MAPK Phosphatase INDOLE-3-BUTYRIC ACID-RESPONSE5 (IBR5) as a Novel Regulator of Organ Size in Arabidopsis.

Johnson KL, Ramm S, Kappel C, Ward S, Leyser O, Sakamoto T, Kurata T, Bevan MW, Lenhard M - PLoS ONE (2015)

Bottom Line: Organ size and shape regulation by IBR5 acts independently of the KLU growth-regulatory pathway.Microarray analysis of tink/ibr5-6 mutants identified a likely role for this phosphatase in male gametophyte development.We show that IBR5 may influence the size and shape of petals through auxin and TCP growth regulatory pathways.

View Article: PubMed Central - PubMed

Affiliation: ARC Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, Royal Parade, Parkville, Victoria, 3010, Australia.

ABSTRACT
Mitogen-activated dual-specificity MAPK phosphatases are important negative regulators in the MAPK signalling pathways responsible for many essential processes in plants. In a screen for mutants with reduced organ size we have identified a mutation in the active site of the dual-specificity MAPK phosphatase indole-3-butyric acid-response5 (IBR5) that we named tinkerbell (tink) due to its small size. Analysis of the tink mutant indicates that IBR5 acts as a novel regulator of organ size that changes the rate of growth in petals and leaves. Organ size and shape regulation by IBR5 acts independently of the KLU growth-regulatory pathway. Microarray analysis of tink/ibr5-6 mutants identified a likely role for this phosphatase in male gametophyte development. We show that IBR5 may influence the size and shape of petals through auxin and TCP growth regulatory pathways.

No MeSH data available.


Related in: MedlinePlus

Identification of the tink/ibr5-6 causal mutation.a. Schematic representation of the IBR5 gene showing point mutation sites of mutants generated by EMS (indicated by arrows), ibr5-1 (causing a premature stop codon), ibr5-4 (G727 to A mutation in the third exon that changes G132 to E), ibr5-5 (G to A mutation in the intron of the last intron-exon junction) and tink/ibr5-6 in the third exon (asterix, G721 to A transition in tink/ibr5-6 changes C129 to Y in the conserved dual-specificity phosphatase catalytic domain), and T-DNA insertion sites of ibr5-2 and ibr5-3 in the promoter and second exon (open triangles). b. Alignment of IBR5 with plant homologues from Arabidopsis lyrata (Al), rice (Os), maize (Zm), Physcomitrella patens (Pp) and animal homologs from Xenopus laevis (Xl) and humans (Hs) showing the conserved phosphatase loop region. Identical residues are shaded in black and similar residues in at least two and four sequences are shaded in light and dark grey respectively. The G to A transition in tink/ibr5-6 that changes the active-site Cysteine residue to a Tyrosine is indicated with an asterisk. c. Petal size measurements of Ler, tink/ibr5-6, and tink/ibr5-6 plants complemented with p35S::GFP:IBR5 or p35S::IBR5 constructs. The significant reduction in size of tink/ibr5-6 petals compared to Ler (shown by *, p value ≤ 2.6e-16, two tailed t-test) is partially rescued in tink1/ibr5-6 GFP:IBR5 and tink1/ibr5-6 IBR5 petals. Petal size of tink1/ibr5-6 GFP:IBR5 and tink1/ibr5-6 IBR5 is significantly larger than that of tink/ibr5-6 (shown by **; p value ≤ 6e-12, IBR5:GFP and p ≤ 1.3e-11, IBR5) in two tailed t-tests assuming unequal variance. Values are shown as mean ± SEM, with n = 20.
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pone.0131103.g002: Identification of the tink/ibr5-6 causal mutation.a. Schematic representation of the IBR5 gene showing point mutation sites of mutants generated by EMS (indicated by arrows), ibr5-1 (causing a premature stop codon), ibr5-4 (G727 to A mutation in the third exon that changes G132 to E), ibr5-5 (G to A mutation in the intron of the last intron-exon junction) and tink/ibr5-6 in the third exon (asterix, G721 to A transition in tink/ibr5-6 changes C129 to Y in the conserved dual-specificity phosphatase catalytic domain), and T-DNA insertion sites of ibr5-2 and ibr5-3 in the promoter and second exon (open triangles). b. Alignment of IBR5 with plant homologues from Arabidopsis lyrata (Al), rice (Os), maize (Zm), Physcomitrella patens (Pp) and animal homologs from Xenopus laevis (Xl) and humans (Hs) showing the conserved phosphatase loop region. Identical residues are shaded in black and similar residues in at least two and four sequences are shaded in light and dark grey respectively. The G to A transition in tink/ibr5-6 that changes the active-site Cysteine residue to a Tyrosine is indicated with an asterisk. c. Petal size measurements of Ler, tink/ibr5-6, and tink/ibr5-6 plants complemented with p35S::GFP:IBR5 or p35S::IBR5 constructs. The significant reduction in size of tink/ibr5-6 petals compared to Ler (shown by *, p value ≤ 2.6e-16, two tailed t-test) is partially rescued in tink1/ibr5-6 GFP:IBR5 and tink1/ibr5-6 IBR5 petals. Petal size of tink1/ibr5-6 GFP:IBR5 and tink1/ibr5-6 IBR5 is significantly larger than that of tink/ibr5-6 (shown by **; p value ≤ 6e-12, IBR5:GFP and p ≤ 1.3e-11, IBR5) in two tailed t-tests assuming unequal variance. Values are shown as mean ± SEM, with n = 20.

Mentions: An F2 population of a backcross of tink mutants to Col-0 wild type was used for whole genome sequencing to identify the causal mutation. Rough mapping and analysis of SNPs distribution (S1C Fig) indicated that the mutation was located on the short arm of Chromosome 2. Further analysis of SNPs within this region revealed a G-to-A transition typical of EMS mutagenesis within the coding region of At2g04550 that was associated with the tink mutant (Fig 2A). At2g04550 corresponds to the previously characterised IBR5 gene that encodes a dual specificity protein phosphatase 1E [8]. Therefore tink represents a new mutant allele of IBR5 that will also be referred to as ibr5-6. Dual specificity protein phosphatases are characterized by a highly conserved active site motif VxVHCx2GxSRSx5AYLM, with the cysteine and arginine residues participating with the conserved aspartate in catalysis [18, 22]. The cysteine of this signature begins the dephosphorylation process with a nucleophilic attack on the phosphorus atom of the phosphotyrosine or phosphothreonine substrate. Disruption of this conserved cysteine has been shown to result in catalytic inactivity [19]. The G-to-A transition in tink/ibr5-6 changes the active cysteine residue to a tyrosine (Fig 2B). Complementation of the tink/ibr5-6 mutant with both p35S::IBR5 and p35S::GFP:IBR5 construct partially recovered wild-type petal size, indicating that loss of phosphatase activity of IBR5 contributes to the tink/ibr5-6 phenotype (Fig 2C).


The Tinkerbell (Tink) Mutation Identifies the Dual-Specificity MAPK Phosphatase INDOLE-3-BUTYRIC ACID-RESPONSE5 (IBR5) as a Novel Regulator of Organ Size in Arabidopsis.

Johnson KL, Ramm S, Kappel C, Ward S, Leyser O, Sakamoto T, Kurata T, Bevan MW, Lenhard M - PLoS ONE (2015)

Identification of the tink/ibr5-6 causal mutation.a. Schematic representation of the IBR5 gene showing point mutation sites of mutants generated by EMS (indicated by arrows), ibr5-1 (causing a premature stop codon), ibr5-4 (G727 to A mutation in the third exon that changes G132 to E), ibr5-5 (G to A mutation in the intron of the last intron-exon junction) and tink/ibr5-6 in the third exon (asterix, G721 to A transition in tink/ibr5-6 changes C129 to Y in the conserved dual-specificity phosphatase catalytic domain), and T-DNA insertion sites of ibr5-2 and ibr5-3 in the promoter and second exon (open triangles). b. Alignment of IBR5 with plant homologues from Arabidopsis lyrata (Al), rice (Os), maize (Zm), Physcomitrella patens (Pp) and animal homologs from Xenopus laevis (Xl) and humans (Hs) showing the conserved phosphatase loop region. Identical residues are shaded in black and similar residues in at least two and four sequences are shaded in light and dark grey respectively. The G to A transition in tink/ibr5-6 that changes the active-site Cysteine residue to a Tyrosine is indicated with an asterisk. c. Petal size measurements of Ler, tink/ibr5-6, and tink/ibr5-6 plants complemented with p35S::GFP:IBR5 or p35S::IBR5 constructs. The significant reduction in size of tink/ibr5-6 petals compared to Ler (shown by *, p value ≤ 2.6e-16, two tailed t-test) is partially rescued in tink1/ibr5-6 GFP:IBR5 and tink1/ibr5-6 IBR5 petals. Petal size of tink1/ibr5-6 GFP:IBR5 and tink1/ibr5-6 IBR5 is significantly larger than that of tink/ibr5-6 (shown by **; p value ≤ 6e-12, IBR5:GFP and p ≤ 1.3e-11, IBR5) in two tailed t-tests assuming unequal variance. Values are shown as mean ± SEM, with n = 20.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4492785&req=5

pone.0131103.g002: Identification of the tink/ibr5-6 causal mutation.a. Schematic representation of the IBR5 gene showing point mutation sites of mutants generated by EMS (indicated by arrows), ibr5-1 (causing a premature stop codon), ibr5-4 (G727 to A mutation in the third exon that changes G132 to E), ibr5-5 (G to A mutation in the intron of the last intron-exon junction) and tink/ibr5-6 in the third exon (asterix, G721 to A transition in tink/ibr5-6 changes C129 to Y in the conserved dual-specificity phosphatase catalytic domain), and T-DNA insertion sites of ibr5-2 and ibr5-3 in the promoter and second exon (open triangles). b. Alignment of IBR5 with plant homologues from Arabidopsis lyrata (Al), rice (Os), maize (Zm), Physcomitrella patens (Pp) and animal homologs from Xenopus laevis (Xl) and humans (Hs) showing the conserved phosphatase loop region. Identical residues are shaded in black and similar residues in at least two and four sequences are shaded in light and dark grey respectively. The G to A transition in tink/ibr5-6 that changes the active-site Cysteine residue to a Tyrosine is indicated with an asterisk. c. Petal size measurements of Ler, tink/ibr5-6, and tink/ibr5-6 plants complemented with p35S::GFP:IBR5 or p35S::IBR5 constructs. The significant reduction in size of tink/ibr5-6 petals compared to Ler (shown by *, p value ≤ 2.6e-16, two tailed t-test) is partially rescued in tink1/ibr5-6 GFP:IBR5 and tink1/ibr5-6 IBR5 petals. Petal size of tink1/ibr5-6 GFP:IBR5 and tink1/ibr5-6 IBR5 is significantly larger than that of tink/ibr5-6 (shown by **; p value ≤ 6e-12, IBR5:GFP and p ≤ 1.3e-11, IBR5) in two tailed t-tests assuming unequal variance. Values are shown as mean ± SEM, with n = 20.
Mentions: An F2 population of a backcross of tink mutants to Col-0 wild type was used for whole genome sequencing to identify the causal mutation. Rough mapping and analysis of SNPs distribution (S1C Fig) indicated that the mutation was located on the short arm of Chromosome 2. Further analysis of SNPs within this region revealed a G-to-A transition typical of EMS mutagenesis within the coding region of At2g04550 that was associated with the tink mutant (Fig 2A). At2g04550 corresponds to the previously characterised IBR5 gene that encodes a dual specificity protein phosphatase 1E [8]. Therefore tink represents a new mutant allele of IBR5 that will also be referred to as ibr5-6. Dual specificity protein phosphatases are characterized by a highly conserved active site motif VxVHCx2GxSRSx5AYLM, with the cysteine and arginine residues participating with the conserved aspartate in catalysis [18, 22]. The cysteine of this signature begins the dephosphorylation process with a nucleophilic attack on the phosphorus atom of the phosphotyrosine or phosphothreonine substrate. Disruption of this conserved cysteine has been shown to result in catalytic inactivity [19]. The G-to-A transition in tink/ibr5-6 changes the active cysteine residue to a tyrosine (Fig 2B). Complementation of the tink/ibr5-6 mutant with both p35S::IBR5 and p35S::GFP:IBR5 construct partially recovered wild-type petal size, indicating that loss of phosphatase activity of IBR5 contributes to the tink/ibr5-6 phenotype (Fig 2C).

Bottom Line: Organ size and shape regulation by IBR5 acts independently of the KLU growth-regulatory pathway.Microarray analysis of tink/ibr5-6 mutants identified a likely role for this phosphatase in male gametophyte development.We show that IBR5 may influence the size and shape of petals through auxin and TCP growth regulatory pathways.

View Article: PubMed Central - PubMed

Affiliation: ARC Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, Royal Parade, Parkville, Victoria, 3010, Australia.

ABSTRACT
Mitogen-activated dual-specificity MAPK phosphatases are important negative regulators in the MAPK signalling pathways responsible for many essential processes in plants. In a screen for mutants with reduced organ size we have identified a mutation in the active site of the dual-specificity MAPK phosphatase indole-3-butyric acid-response5 (IBR5) that we named tinkerbell (tink) due to its small size. Analysis of the tink mutant indicates that IBR5 acts as a novel regulator of organ size that changes the rate of growth in petals and leaves. Organ size and shape regulation by IBR5 acts independently of the KLU growth-regulatory pathway. Microarray analysis of tink/ibr5-6 mutants identified a likely role for this phosphatase in male gametophyte development. We show that IBR5 may influence the size and shape of petals through auxin and TCP growth regulatory pathways.

No MeSH data available.


Related in: MedlinePlus