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The M3 phosphorylation motif has been functionally conserved for intracellular trafficking of long-looped PIN-FORMEDs in the Arabidopsis root hair cell.

Sasayama D, Ganguly A, Park M, Cho HT - BMC Plant Biol. (2013)

Bottom Line: Root hair-specific overexpression of wild-type PIN1, 2, or 7 greatly inhibited root hair growth by depleting auxin levels in the root hair cell, whereas overexpression of M3 phosphorylation-defective PIN mutants failed to inhibit root hair growth.Consistent with this root hair phenotype, the PM localization of M3 phosphorylation-defective PIN1 and PIN7 was partially disrupted, resulting in less auxin efflux and restoration of root hair growth.On the other hand, compared with the PIN1 and PIN7 mutant proteins, M3-phosphorylation-defective PIN2 proteins were almost undetectable.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences and Plant Genomics and Breeding Institute, Seoul National University, Seoul 151-742, Korea. htcho@snu.ac.kr.

ABSTRACT

Background: PIN-FORMED (PIN) efflux carriers contribute to polar auxin transport and plant development by exhibiting dynamic and diverse asymmetrical localization patterns in the plasma membrane (PM). Phosphorylation of the central hydrophilic loop (HL) of PINs has been implicated in the regulation of PIN trafficking. Recently, we reported that a phosphorylatable motif (M3) in the PIN3-HL is necessary for the polarity, intracellular trafficking, and biological functions of PIN3. In this study, using the root hair system for PIN activity assay, we investigated whether this motif has been functionally conserved among long-HL PINs.

Results: Root hair-specific overexpression of wild-type PIN1, 2, or 7 greatly inhibited root hair growth by depleting auxin levels in the root hair cell, whereas overexpression of M3 phosphorylation-defective PIN mutants failed to inhibit root hair growth. Consistent with this root hair phenotype, the PM localization of M3 phosphorylation-defective PIN1 and PIN7 was partially disrupted, resulting in less auxin efflux and restoration of root hair growth. Partial formation of brefeldin A-compartments in these phosphorylation-mutant PIN lines also suggested that their PM targeting was partially disrupted. On the other hand, compared with the PIN1 and PIN7 mutant proteins, M3-phosphorylation-defective PIN2 proteins were almost undetectable. However, the mutant PIN2 protein levels were restored by wortmannin treatment almost to the wild-type PIN2 level, indicating that the M3 motif of PIN2, unlike that of other PINs, is implicated in PIN2 trafficking to the vacuolar lytic pathway.

Conclusions: These results suggest that the M3 phosphorylation motif has been functionally conserved to modulate the intracellular trafficking of long-HL PINs, but its specific function in trafficking has diverged among PIN members.

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Related in: MedlinePlus

M3 mutations greatly decreased cellular PIN2 levels in root hair cells. Confocal images showing the subcellular localization of ProE7:PIN2 (WT-PIN2), ProE7:3 m1-PIN2, and proE7:M3-PIN2 in root hair cells. Transgenic seedlings were stained with FM4-64 (2 μM). Representative images are shown. Bar = 10 μm in all cases.
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Figure 4: M3 mutations greatly decreased cellular PIN2 levels in root hair cells. Confocal images showing the subcellular localization of ProE7:PIN2 (WT-PIN2), ProE7:3 m1-PIN2, and proE7:M3-PIN2 in root hair cells. Transgenic seedlings were stained with FM4-64 (2 μM). Representative images are shown. Bar = 10 μm in all cases.

Mentions: Unlike phosphorylation-defective PIN1 and PIN7, the mutant PIN2 proteins showed completely different trafficking behavior. While wild-type PIN2 was clearly localized to the PM, phosphorylation-defective 3 m1- or M3-PIN2 proteins were barely detectable in root hair cells; this was consistent in all five independent transgenic lines observed for each mutant construct (Figure 4 and Additional file 1: Figure S6A). This result was in accordance with the results from the root-hair-inhibition assay, where 3 m1 or M3 mutant PIN2s showed much weaker inhibitory effects (~30% and ~15% inhibitory effects for 3 m1 and M3, respectively) on root hair growth than did mutant PIN1 or PIN7 (Figure 1B).


The M3 phosphorylation motif has been functionally conserved for intracellular trafficking of long-looped PIN-FORMEDs in the Arabidopsis root hair cell.

Sasayama D, Ganguly A, Park M, Cho HT - BMC Plant Biol. (2013)

M3 mutations greatly decreased cellular PIN2 levels in root hair cells. Confocal images showing the subcellular localization of ProE7:PIN2 (WT-PIN2), ProE7:3 m1-PIN2, and proE7:M3-PIN2 in root hair cells. Transgenic seedlings were stained with FM4-64 (2 μM). Representative images are shown. Bar = 10 μm in all cases.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222813&req=5

Figure 4: M3 mutations greatly decreased cellular PIN2 levels in root hair cells. Confocal images showing the subcellular localization of ProE7:PIN2 (WT-PIN2), ProE7:3 m1-PIN2, and proE7:M3-PIN2 in root hair cells. Transgenic seedlings were stained with FM4-64 (2 μM). Representative images are shown. Bar = 10 μm in all cases.
Mentions: Unlike phosphorylation-defective PIN1 and PIN7, the mutant PIN2 proteins showed completely different trafficking behavior. While wild-type PIN2 was clearly localized to the PM, phosphorylation-defective 3 m1- or M3-PIN2 proteins were barely detectable in root hair cells; this was consistent in all five independent transgenic lines observed for each mutant construct (Figure 4 and Additional file 1: Figure S6A). This result was in accordance with the results from the root-hair-inhibition assay, where 3 m1 or M3 mutant PIN2s showed much weaker inhibitory effects (~30% and ~15% inhibitory effects for 3 m1 and M3, respectively) on root hair growth than did mutant PIN1 or PIN7 (Figure 1B).

Bottom Line: Root hair-specific overexpression of wild-type PIN1, 2, or 7 greatly inhibited root hair growth by depleting auxin levels in the root hair cell, whereas overexpression of M3 phosphorylation-defective PIN mutants failed to inhibit root hair growth.Consistent with this root hair phenotype, the PM localization of M3 phosphorylation-defective PIN1 and PIN7 was partially disrupted, resulting in less auxin efflux and restoration of root hair growth.On the other hand, compared with the PIN1 and PIN7 mutant proteins, M3-phosphorylation-defective PIN2 proteins were almost undetectable.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences and Plant Genomics and Breeding Institute, Seoul National University, Seoul 151-742, Korea. htcho@snu.ac.kr.

ABSTRACT

Background: PIN-FORMED (PIN) efflux carriers contribute to polar auxin transport and plant development by exhibiting dynamic and diverse asymmetrical localization patterns in the plasma membrane (PM). Phosphorylation of the central hydrophilic loop (HL) of PINs has been implicated in the regulation of PIN trafficking. Recently, we reported that a phosphorylatable motif (M3) in the PIN3-HL is necessary for the polarity, intracellular trafficking, and biological functions of PIN3. In this study, using the root hair system for PIN activity assay, we investigated whether this motif has been functionally conserved among long-HL PINs.

Results: Root hair-specific overexpression of wild-type PIN1, 2, or 7 greatly inhibited root hair growth by depleting auxin levels in the root hair cell, whereas overexpression of M3 phosphorylation-defective PIN mutants failed to inhibit root hair growth. Consistent with this root hair phenotype, the PM localization of M3 phosphorylation-defective PIN1 and PIN7 was partially disrupted, resulting in less auxin efflux and restoration of root hair growth. Partial formation of brefeldin A-compartments in these phosphorylation-mutant PIN lines also suggested that their PM targeting was partially disrupted. On the other hand, compared with the PIN1 and PIN7 mutant proteins, M3-phosphorylation-defective PIN2 proteins were almost undetectable. However, the mutant PIN2 protein levels were restored by wortmannin treatment almost to the wild-type PIN2 level, indicating that the M3 motif of PIN2, unlike that of other PINs, is implicated in PIN2 trafficking to the vacuolar lytic pathway.

Conclusions: These results suggest that the M3 phosphorylation motif has been functionally conserved to modulate the intracellular trafficking of long-HL PINs, but its specific function in trafficking has diverged among PIN members.

Show MeSH
Related in: MedlinePlus