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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

Investigation of auxin pathways in tink/ibr5-6 mutants.a. Real time quantitative (Q)-PCR validation (upper panel) of genes implicated in auxin biogenesis or transport identified in microarray analysis (lower panel) as being altered in tink/ibr5-6 mutants compared to wild-type flowers. Values in Q-PCR analysis are shown as mean ± SEM with expression levels normalized to that of the TUB6 gene for 3 biological and 3 technical replicates. b. Quantitation of bulk polar IAA transport in stem segments of the indicated genotypes. Values are represented as mean ± SEM of radiolabel transported (in cpm). There is no significant difference between wild-type and the respective ibr5 mutant alleles. At least 18 stem segments were assayed per genotype.
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pone.0131103.g005: Investigation of auxin pathways in tink/ibr5-6 mutants.a. Real time quantitative (Q)-PCR validation (upper panel) of genes implicated in auxin biogenesis or transport identified in microarray analysis (lower panel) as being altered in tink/ibr5-6 mutants compared to wild-type flowers. Values in Q-PCR analysis are shown as mean ± SEM with expression levels normalized to that of the TUB6 gene for 3 biological and 3 technical replicates. b. Quantitation of bulk polar IAA transport in stem segments of the indicated genotypes. Values are represented as mean ± SEM of radiolabel transported (in cpm). There is no significant difference between wild-type and the respective ibr5 mutant alleles. At least 18 stem segments were assayed per genotype.

Mentions: Given the previous association of IBR5 with regulation of auxin pathways and results showing reduced expression of pDR5:GUS in the ibr5-1 mutant [8], we were interested to know if auxin responsive genes are mis-regulated in tink/ibr5-6. A set of genes identified in microarray studies as being significantly up- or down-regulated in tink/ibr5-6 compared to wild-type were validated by Q-PCR. These genes are annotated as being involved in auxin synthesis, transport, regulation and responses (S2 Table, Fig 5A). Intriguingly this list included a number of genes involved in auxin efflux (PINs). This supports previous studies that IBR5 is involved in auxin signalling pathways. Auxin transport assays performed on basal stem segments of ibr5-3 and tink/ibr5-6 mutants compared to the respective wild-types showed no significant differences (Fig 5B). To determine if changes in auxin distribution are responsible for the altered petal phenotype, the pDR5:GFP reporter was introduced into tink/ibr5-6 and ibr5-3 mutants [9]. In wild-type petals pDR5:GFP is localized at the tip of developing petals and in the vasculature [26]. No difference to wild-type pDR5:GFP pattern of expression was observed in the ibr5 mutant lines (S4 Fig). A reduction in the level of pDR5:GUS in roots and leaves has previously been observed in ibr5-1 mutants, even in tissues where pIBR5:GUS was not observed [8, 19]. From our investigation of pDR5:GFP expression in petals of ibr5 mutants, either no change in auxin levels occurs or our methods are not sensitive enough to detect potentially subtle changes in pDR5:GFP expression.


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)

Investigation of auxin pathways in tink/ibr5-6 mutants.a. Real time quantitative (Q)-PCR validation (upper panel) of genes implicated in auxin biogenesis or transport identified in microarray analysis (lower panel) as being altered in tink/ibr5-6 mutants compared to wild-type flowers. Values in Q-PCR analysis are shown as mean ± SEM with expression levels normalized to that of the TUB6 gene for 3 biological and 3 technical replicates. b. Quantitation of bulk polar IAA transport in stem segments of the indicated genotypes. Values are represented as mean ± SEM of radiolabel transported (in cpm). There is no significant difference between wild-type and the respective ibr5 mutant alleles. At least 18 stem segments were assayed per genotype.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131103.g005: Investigation of auxin pathways in tink/ibr5-6 mutants.a. Real time quantitative (Q)-PCR validation (upper panel) of genes implicated in auxin biogenesis or transport identified in microarray analysis (lower panel) as being altered in tink/ibr5-6 mutants compared to wild-type flowers. Values in Q-PCR analysis are shown as mean ± SEM with expression levels normalized to that of the TUB6 gene for 3 biological and 3 technical replicates. b. Quantitation of bulk polar IAA transport in stem segments of the indicated genotypes. Values are represented as mean ± SEM of radiolabel transported (in cpm). There is no significant difference between wild-type and the respective ibr5 mutant alleles. At least 18 stem segments were assayed per genotype.
Mentions: Given the previous association of IBR5 with regulation of auxin pathways and results showing reduced expression of pDR5:GUS in the ibr5-1 mutant [8], we were interested to know if auxin responsive genes are mis-regulated in tink/ibr5-6. A set of genes identified in microarray studies as being significantly up- or down-regulated in tink/ibr5-6 compared to wild-type were validated by Q-PCR. These genes are annotated as being involved in auxin synthesis, transport, regulation and responses (S2 Table, Fig 5A). Intriguingly this list included a number of genes involved in auxin efflux (PINs). This supports previous studies that IBR5 is involved in auxin signalling pathways. Auxin transport assays performed on basal stem segments of ibr5-3 and tink/ibr5-6 mutants compared to the respective wild-types showed no significant differences (Fig 5B). To determine if changes in auxin distribution are responsible for the altered petal phenotype, the pDR5:GFP reporter was introduced into tink/ibr5-6 and ibr5-3 mutants [9]. In wild-type petals pDR5:GFP is localized at the tip of developing petals and in the vasculature [26]. No difference to wild-type pDR5:GFP pattern of expression was observed in the ibr5 mutant lines (S4 Fig). A reduction in the level of pDR5:GUS in roots and leaves has previously been observed in ibr5-1 mutants, even in tissues where pIBR5:GUS was not observed [8, 19]. From our investigation of pDR5:GFP expression in petals of ibr5 mutants, either no change in auxin levels occurs or our methods are not sensitive enough to detect potentially subtle changes in pDR5:GFP expression.

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