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

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.

Figure 1

2-BP abolished the tonoplast localization of CBL2 in root hairs. A-D. 4 DAG seedlings of Pro _UBQ10:CBL2-RFP transgenic plants treated with either DMSO (A, B) or 2-BP (C, D) for 4–12 hr before visualization. E-F. 4 DAG seedlings of Pro _UBQ10:CBL2-RFP;pat10-2 transgenic plants treated with DMSO for 4–12 hr before imaging. Representative initiating (A, C, E) or elongating (B, D, F) root hairs are shown. V indicates vacuole. G. Quantification of CBL2-RFP distribution in the tonoplast v.s. the cytoplasm (Tonoplast/Cyt) at different time points after 2-BP treatment. a.u. stands for arbitrary fluorescence units. Bars = 7.5 μm.

Figure 2

2-BP impaired root hair growth. A. Primary roots from 4 DAG seedlings of wild type treated with either DMSO or 2-BP. B. Primary roots from 4 DAG seedlings of pat10-2 treated with either DMSO or 2-BP. C. Primary roots from 4 DAG seedlings of tip1-4 treated with either DMSO or 2-BP. D-E. Representative root hairs at the hair elongation zone of 4 DAG seedlings treated with either DMSO (D) or with 2-BP (E). F-G. Root hair length (F) and width (G). Results are means ± standard errors (SE), N = 4. Length or width of mature wild-type root hairs treated with DMSO was set as 1. Empty bars represent DMSO treatment while filled bars represent 2-BP treatment. Asterisks indicate significant difference (Student’s t-test, P < 0.05). Bars = 500 μm for (A-C); 20 μm for (D-E).

Figure 3

TIP1 and PAT10 localize at different endomembrane compartments in root hairs. A-C. Representative initiating root hair (A), elongating root hair (B), or mature root hair (C) of 4 DAG PAT10g-GFP;pat10-2 transgenic seedlings. D-F. Representative initiating root hair (D), elongating root hair (E), or mature root hair (F) of 4 DAG TIP1g-GFP;tip1-4 transgenic seedlings. Bars = 10 μm.

Figure 4

2-BP induced fragmentation and cross-linking of actin MF in root hairs. 4 DAG seedlings of Pro _35S:GFP-ABD2-GFP transgenic seedlings were treated with DMSO or with 10 μM 2-BP for 2–3 hr before imaging. 18 to 20 root hairs at different stages were examined and representative images are shown. Single section indicates one optical section taken at the mid-plane of a root hair. For each root hair shown, twenty 1 μm optical sections were superimposed to generate the projection of Z-stacks. Bars = 10 μm.

Figure 5

2-BP treatment re-distributed the PIP ₂ sensor 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.

Figure 6

2-BP treatment relocalizes ROP2 from the PM to the cytoplasm. A-C. Representative initiating root hair (A), elongating root hair (B), or mature root hair (C) of 4 DAG Pro _E7: GFP-ROP2 transgenic seedlings treated with DMSO for 3 hr. D-F. Representative initiating root hair (D), elongating root hair (E), or mature root hair (F) of 4 DAG Pro _E7: GFP-ROP2 transgenic seedlings treated with 2-BP for 3 hr. G. Ratio of fluorescence signal intensity indicating the relative distribution of ROP2 in the cytoplasm and PM (Cyt/PM). a.u. stands for arbitrary fluorescence units. Results are means ± SD, n = 16. Bars = 7.5 μm.

Figure 7

2-BP treatment interfered with the dynamics of RabA4b-positive secretory vesicles. A. Distribution of RabA4b-positive vesicles in a growing root hair of 4 DAG RFP-RabA4b transgenic seedlings treated with DMSO for 2 hr. The arrowhead points at the base of the clear zone where RabA4b-positive vesicles form an inverted cone. Below is merge of fluorescence and bright field images. B-D. Distribution of RabA4b-positive vesicles in a root hair right after initiation (B), a growing root hair (C), or an arrested root hair (D) of 4 DAG RFP-RabA4b transgenic seedlings treated with 10 μM 2-BP for 2 hr. The arrows point at enlarged vesicles positive for RabA4b. Below are the merges of fluorescence and bright field images. E-F. RFP-RabA4b fluorescence was visualized in root hairs treated with DMSO (E) or 10 μM 2-BP (F) for 2 hr using time-lapse confocal fluorescence microscopy. Left-most are the bright-field images. The arrowhead points to the base of the clear-zone. The arrows follow the moving track of a single large vesicle over time. Bars = 7.5 μm.

Figure 8

2-BP inhibited endocytosis in root hairs. A. FM4-64 uptake in initiating root hairs pre-treated with DMSO for 2 hr. B. FM4-64 uptake in initiating root hairs pre-treated with 10 μM 2-BP for 2 hr. C. FM4-64 uptake in elongating root hairs pre-treated with DMSO for 2 hr. D. FM4-64 uptake in elongating root hairs pre-treated with 10 μM 2-BP for 2 hr. Images shown are representative of 18–25 root hairs analyzed in three independent experiments. Bars = 10 μm.

Figure 9

2-BP interfered with vacuolar trafficking. A-D. 4 DAG WT seedlings were pulse-labeled with FM4-64, washed and incubated for 30 min (A), or pulse-labeled with FM4-64 followed by 30 min incubation with 1/2 MS medium supplemented with 50 μM BFA (B). BFA-treated seedlings were then washed with 1/2 MS medium supplemented with either DMSO (BFA wo + DMSO) (C) or 2-BP (BFA wo + 2-BP) (D). Arrows point at the tonoplat labeled by FM4-64. Arrowhead indicates the BFA compartment. Results are representative of 20 root hairs for each treatment. Bars = 7.5 μm.