Limits...
Protein palmitoylation is critical for the polar growth of root hairs in Arabidopsis.

Zhang YL, Li E, Feng QN, Zhao XY, Ge FR, Zhang Y, Li S - BMC Plant Biol. (2015)

Bottom Line: By combining pharmacological and genetic approaches and using root hairs as a model, we show that protein palmitoylation, regulated by protein S-acyl transferases at different endomembrane compartments such as the Golgi and the vacuole, is critical for the polar growth of root hairs in Arabidopsis.Inhibition of protein palmitoylation by 2-BP disturbed key intracellular activities in root hairs.Although some of these effects are likely indirect, the cytological data reported here will contribute to a deep understanding of protein palmitoylation during tip growth in plants.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Protein palmitoylation, which is critical for membrane association and subcellular targeting of many signaling proteins, is catalyzed mainly by protein S-acyl transferases (PATs). Only a few plant proteins have been experimentally verified to be subject to palmitoylation, such as ROP GTPases, calcineurin B like proteins (CBLs), and subunits of heterotrimeric G proteins. However, emerging evidence from palmitoyl proteomics hinted that protein palmitoylation as a post-translational modification might be widespread. Nonetheless, due to the large number of genes encoding PATs and the lack of consensus motifs for palmitoylation, progress on the roles of protein palmitoylation in plants has been slow.

Results: We combined pharmacological and genetic approaches to examine the role of protein palmitoylation in root hair growth. Multiple PATs from different endomembrane compartments may participate in root hair growth, among which the Golgi-localized PAT24/TIP GROWTH DEFECTIVE1 (TIP1) plays a major role while the tonoplast-localized PAT10 plays a secondary role in root hair growth. A specific inhibitor for protein palmitoylation, 2-bromopalmitate (2-BP), compromised root hair elongation and polarity. Using various probes specific for cellular processes, we demonstrated that 2-BP impaired the dynamic polymerization of actin microfilaments (MF), the asymmetric plasma membrane (PM) localization of phosphatidylinositol (4,5)-bisphosphate (PIP2), the dynamic distribution of RabA4b-positive post-Golgi secretion, and endocytic trafficking in root hairs.

Conclusions: By combining pharmacological and genetic approaches and using root hairs as a model, we show that protein palmitoylation, regulated by protein S-acyl transferases at different endomembrane compartments such as the Golgi and the vacuole, is critical for the polar growth of root hairs in Arabidopsis. Inhibition of protein palmitoylation by 2-BP disturbed key intracellular activities in root hairs. Although some of these effects are likely indirect, the cytological data reported here will contribute to a deep understanding of protein palmitoylation during tip growth in plants.

Show MeSH

Related in: MedlinePlus

2-BP treatment re-distributed the PIP2sensor from the PM to the cytoplasm in root hairs. A. DMSO-treated root hairs expressing the PIP2 sensor (green) at the initiating stage. B. 2-BP-treated root hairs expressing the PIP2 sensor at the initiating stage. C. DMSO-treated root hairs expressing the PIP2 sensor at the elongating stage. D. 2-BP-treated root hairs expressing the PIP2 sensor at the elongating stage. E. Ratio of fluorescence signals. a.u. stands for arbitrary fluorescence units. Cyt/PM indicates the ratio of cytoplasmic to the plasma membrane signal. Results are means ± standard deviation (SD, n = 30). Asterisk indicates significant difference (Student’s t-test, P < 0.01). Root hairs were stained with the fluorescence dye propidium iodide (red) to outline cell shape. Corresponding bright-field images are shown together with merges of different channels. Bars = 7.5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4340681&req=5

Fig5: 2-BP treatment re-distributed the PIP2sensor from the PM to the cytoplasm in root hairs. A. DMSO-treated root hairs expressing the PIP2 sensor (green) at the initiating stage. B. 2-BP-treated root hairs expressing the PIP2 sensor at the initiating stage. C. DMSO-treated root hairs expressing the PIP2 sensor at the elongating stage. D. 2-BP-treated root hairs expressing the PIP2 sensor at the elongating stage. E. Ratio of fluorescence signals. a.u. stands for arbitrary fluorescence units. Cyt/PM indicates the ratio of cytoplasmic to the plasma membrane signal. Results are means ± standard deviation (SD, n = 30). Asterisk indicates significant difference (Student’s t-test, P < 0.01). Root hairs were stained with the fluorescence dye propidium iodide (red) to outline cell shape. Corresponding bright-field images are shown together with merges of different channels. Bars = 7.5 μm.

Mentions: To determine the effect of 2-BP on the asymmetric PM localization of PIP2, we treated a P15Y fluorescence sensor line [41] with 2-BP or with DMSO. The P15Y sensor line expresses a ProUBQ10-driven PIP2-binding TUBBY-C fused with CITRINE [41]. As shown by other PIP2 sensors [42], PIP2 was asymmetrically localized at the PM of initiation sites in trichoblasts (Figure 5A). During hair elongation, PIP2 maintained its asymmetric PM localization at the apical region (Figure 5B). Application of 2-BP significantly redistributed fluorescence signals from the PM to cytosol (Figure 5C,D,E), suggesting abolished PIP2 at the PM. For root hairs either at the initiation stage (Figure 5C) or at the elongating stage (Figure 5D), PIP2 was detected mostly in the cytoplasm and hardly at all at the PM. The residual signals at the PM were uniform (Figure 5D) rather than asymmetric (Figure 5B).Figure 5


Protein palmitoylation is critical for the polar growth of root hairs in Arabidopsis.

Zhang YL, Li E, Feng QN, Zhao XY, Ge FR, Zhang Y, Li S - BMC Plant Biol. (2015)

2-BP treatment re-distributed the PIP2sensor from the PM to the cytoplasm in root hairs. A. DMSO-treated root hairs expressing the PIP2 sensor (green) at the initiating stage. B. 2-BP-treated root hairs expressing the PIP2 sensor at the initiating stage. C. DMSO-treated root hairs expressing the PIP2 sensor at the elongating stage. D. 2-BP-treated root hairs expressing the PIP2 sensor at the elongating stage. E. Ratio of fluorescence signals. a.u. stands for arbitrary fluorescence units. Cyt/PM indicates the ratio of cytoplasmic to the plasma membrane signal. Results are means ± standard deviation (SD, n = 30). Asterisk indicates significant difference (Student’s t-test, P < 0.01). Root hairs were stained with the fluorescence dye propidium iodide (red) to outline cell shape. Corresponding bright-field images are shown together with merges of different channels. Bars = 7.5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4340681&req=5

Fig5: 2-BP treatment re-distributed the PIP2sensor from the PM to the cytoplasm in root hairs. A. DMSO-treated root hairs expressing the PIP2 sensor (green) at the initiating stage. B. 2-BP-treated root hairs expressing the PIP2 sensor at the initiating stage. C. DMSO-treated root hairs expressing the PIP2 sensor at the elongating stage. D. 2-BP-treated root hairs expressing the PIP2 sensor at the elongating stage. E. Ratio of fluorescence signals. a.u. stands for arbitrary fluorescence units. Cyt/PM indicates the ratio of cytoplasmic to the plasma membrane signal. Results are means ± standard deviation (SD, n = 30). Asterisk indicates significant difference (Student’s t-test, P < 0.01). Root hairs were stained with the fluorescence dye propidium iodide (red) to outline cell shape. Corresponding bright-field images are shown together with merges of different channels. Bars = 7.5 μm.
Mentions: To determine the effect of 2-BP on the asymmetric PM localization of PIP2, we treated a P15Y fluorescence sensor line [41] with 2-BP or with DMSO. The P15Y sensor line expresses a ProUBQ10-driven PIP2-binding TUBBY-C fused with CITRINE [41]. As shown by other PIP2 sensors [42], PIP2 was asymmetrically localized at the PM of initiation sites in trichoblasts (Figure 5A). During hair elongation, PIP2 maintained its asymmetric PM localization at the apical region (Figure 5B). Application of 2-BP significantly redistributed fluorescence signals from the PM to cytosol (Figure 5C,D,E), suggesting abolished PIP2 at the PM. For root hairs either at the initiation stage (Figure 5C) or at the elongating stage (Figure 5D), PIP2 was detected mostly in the cytoplasm and hardly at all at the PM. The residual signals at the PM were uniform (Figure 5D) rather than asymmetric (Figure 5B).Figure 5

Bottom Line: By combining pharmacological and genetic approaches and using root hairs as a model, we show that protein palmitoylation, regulated by protein S-acyl transferases at different endomembrane compartments such as the Golgi and the vacuole, is critical for the polar growth of root hairs in Arabidopsis.Inhibition of protein palmitoylation by 2-BP disturbed key intracellular activities in root hairs.Although some of these effects are likely indirect, the cytological data reported here will contribute to a deep understanding of protein palmitoylation during tip growth in plants.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Protein palmitoylation, which is critical for membrane association and subcellular targeting of many signaling proteins, is catalyzed mainly by protein S-acyl transferases (PATs). Only a few plant proteins have been experimentally verified to be subject to palmitoylation, such as ROP GTPases, calcineurin B like proteins (CBLs), and subunits of heterotrimeric G proteins. However, emerging evidence from palmitoyl proteomics hinted that protein palmitoylation as a post-translational modification might be widespread. Nonetheless, due to the large number of genes encoding PATs and the lack of consensus motifs for palmitoylation, progress on the roles of protein palmitoylation in plants has been slow.

Results: We combined pharmacological and genetic approaches to examine the role of protein palmitoylation in root hair growth. Multiple PATs from different endomembrane compartments may participate in root hair growth, among which the Golgi-localized PAT24/TIP GROWTH DEFECTIVE1 (TIP1) plays a major role while the tonoplast-localized PAT10 plays a secondary role in root hair growth. A specific inhibitor for protein palmitoylation, 2-bromopalmitate (2-BP), compromised root hair elongation and polarity. Using various probes specific for cellular processes, we demonstrated that 2-BP impaired the dynamic polymerization of actin microfilaments (MF), the asymmetric plasma membrane (PM) localization of phosphatidylinositol (4,5)-bisphosphate (PIP2), the dynamic distribution of RabA4b-positive post-Golgi secretion, and endocytic trafficking in root hairs.

Conclusions: By combining pharmacological and genetic approaches and using root hairs as a model, we show that protein palmitoylation, regulated by protein S-acyl transferases at different endomembrane compartments such as the Golgi and the vacuole, is critical for the polar growth of root hairs in Arabidopsis. Inhibition of protein palmitoylation by 2-BP disturbed key intracellular activities in root hairs. Although some of these effects are likely indirect, the cytological data reported here will contribute to a deep understanding of protein palmitoylation during tip growth in plants.

Show MeSH
Related in: MedlinePlus