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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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M3 motif is required for PIN activities in root hair cells. (A) Representative root images of the control (ProE7:YFP), wild-type PIN expression lines (WT), and phosphorylation-defective PIN expression lines (3 m1 and M3). WT-, 3 m1-, and M3-PINs were expressed under the root hair-specific EXPASIN A7 promoter (ProE7). Bar = 0.1 mm for all. (B) Root hair lengths of PIN-expressing transformants. The results were obtained from 22–59 seedlings/414–2332 root hairs from five independent lines. Data represent means ± SE.
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Figure 1: M3 motif is required for PIN activities in root hair cells. (A) Representative root images of the control (ProE7:YFP), wild-type PIN expression lines (WT), and phosphorylation-defective PIN expression lines (3 m1 and M3). WT-, 3 m1-, and M3-PINs were expressed under the root hair-specific EXPASIN A7 promoter (ProE7). Bar = 0.1 mm for all. (B) Root hair lengths of PIN-expressing transformants. The results were obtained from 22–59 seedlings/414–2332 root hairs from five independent lines. Data represent means ± SE.

Mentions: Consistent with our previous report [3], root hair-specific overexpression of wild-type PIN1, PIN2, or PIN7 greatly inhibited the root hair growth of 4-d-old seedlings by depleting auxin levels in the root hair cell (Figure 1). On the other hand, this PIN-mediated root hair inhibition was significantly suppressed by 3 m1 or M3 mutation, though M3 mutation consistently showed a greater restoration of root hair growth than did 3 m1 mutation (Figure 1), similarly to previous results with PIN3 [25]. Among the three PINs, the 3 m1 and M3 mutants of PIN2 showed the greatest restoration of root hair growth (Figure 1B). These results were reproducible with 15 independent transgenic lines for each PIN (Additional file 1: Figure S2).


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 motif is required for PIN activities in root hair cells. (A) Representative root images of the control (ProE7:YFP), wild-type PIN expression lines (WT), and phosphorylation-defective PIN expression lines (3 m1 and M3). WT-, 3 m1-, and M3-PINs were expressed under the root hair-specific EXPASIN A7 promoter (ProE7). Bar = 0.1 mm for all. (B) Root hair lengths of PIN-expressing transformants. The results were obtained from 22–59 seedlings/414–2332 root hairs from five independent lines. Data represent means ± SE.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: M3 motif is required for PIN activities in root hair cells. (A) Representative root images of the control (ProE7:YFP), wild-type PIN expression lines (WT), and phosphorylation-defective PIN expression lines (3 m1 and M3). WT-, 3 m1-, and M3-PINs were expressed under the root hair-specific EXPASIN A7 promoter (ProE7). Bar = 0.1 mm for all. (B) Root hair lengths of PIN-expressing transformants. The results were obtained from 22–59 seedlings/414–2332 root hairs from five independent lines. Data represent means ± SE.
Mentions: Consistent with our previous report [3], root hair-specific overexpression of wild-type PIN1, PIN2, or PIN7 greatly inhibited the root hair growth of 4-d-old seedlings by depleting auxin levels in the root hair cell (Figure 1). On the other hand, this PIN-mediated root hair inhibition was significantly suppressed by 3 m1 or M3 mutation, though M3 mutation consistently showed a greater restoration of root hair growth than did 3 m1 mutation (Figure 1), similarly to previous results with PIN3 [25]. Among the three PINs, the 3 m1 and M3 mutants of PIN2 showed the greatest restoration of root hair growth (Figure 1B). These results were reproducible with 15 independent transgenic lines for each PIN (Additional file 1: Figure S2).

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