Actin depolymerizing factor ADF7 inhibits actin bundling protein VILLIN1 to regulate root hair formation in response to osmotic stress in Arabidopsis

Abstract

Actin cytoskeleton is essential for root hair formation. However, the underlying molecular mechanisms of actin dynamics in root hair formation in response to abiotic stress are largely undiscovered. Here, genetic analysis showed that actin-depolymerizing protein ADF7 and actin-bundling protein VILLIN1 (VLN1) were positively and negatively involved in root hair formation of Arabidopsis respectively. Moreover, RT-qPCR, GUS staining, western blotting, and genetic analysis revealed that ADF7 played an important role in inhibiting the expression and function of VLN1 during root hair formation. Filament actin (F-actin) dynamics observation and actin pharmacological experiments indicated that ADF7-inhibited-VLN1 pathway led to the decline of F-actin bundling and thick bundle formation, as well as the increase of F-actin depolymerization and turnover to promote root hair formation. Furthermore, the F-actin dynamics mediated by ADF7-inhibited-VLN1 pathway was associated with the reactive oxygen species (ROS) accumulation in root hair formation. Finally, ADF7-inhibited-VLN1 pathway was critical for osmotic stress-induced root hair formation. Our work demonstrates that ADF7 inhibits VLN1 to regulate F-actin dynamics in root hair formation in response to osmotic stress, providing the novel evidence on the F-actin dynamics and their molecular mechanisms in root hair formation and in abiotic stress.

Fig 1. Actin depolymerization protein ADF7 is positively involved in root hair formation and actin bunding protein VLN1 is negatively involved in root hair formation.

(A) Images of root hairs from wild type (Col-0), adf7-2, ADF7 comp #2, ADF7 comp #7, ADF7 OE #13, and ADF7 OE #14. Scale bar, 200 μm. OE, overexpression. (B) Histogram depicting root hair number in (A). (C) Quantification of the percentage of root hairs from H and N cells and root hair formation position in H cells in (A). Values given are means ± SD. (D) Images of root hairs from Col-0, vln1-1, vln1-2, VLN1 comp #9, VLN1 comp #14, VLN1 OE #7, and VLN1 OE #8. Scale bar, 200 μm. (E) Histogram depicting root hair number in (D). (F) Quantification of the percentage of root hairs from H and N cells and root hair formation position in H cells in (D). Values given are means ± SD. Significant difference (P< 0.05) indicated by different letters among genotypes is determined for each condition by one-way ANOVA followed by Tukey’s test in (B), (C), (E), and (F).

Fig 2. ADF7 inhibits the expression of function VLN1 in root hair formation.

(A) RT-qPCR quantification of VLN1 expression level in Col-0, adf7-2, ADF7 comp #2, ADF7 comp #7, ADF7 OE #13, and ADF7 OE #14. (B) RT-qPCR quantification of ADF7 expression level in Col-0, vln1-1, vln1-2, VLN1 comp #9, VLN1 comp #14, VLN1 OE #7, and VLN1 OE #8. (C) GUS analysis of VLN1 expression in root tips from Col-0, adf7-2, and ADF7 OE seedlings. (D) GUS analysis of ADF7 expression in root tips from Col-0, vln1, and VLN1 OE seedlings. (E) Western blotting of VLN1 expression level in Col-0, adf7-2, and ADF7 OE seedlings. Rubisco as a loading control. (F) Quantification of the relative grayscale value in (E). (G) Western blotting of ADF7 expression level in Col-0, vln1, and VLN1 OE seedlings. Rubisco as a loading control. (H) Quantification of the relative grayscale value in (G). (I) Images of root hairs from ADF7 and VLN1 double gene genotypes. Scale bar, 200 μm. (J) Histogram depicting root hair number in (I). Significant difference (P< 0.05) indicated by different letters among different genotypes is determined for each condition by one-way ANOVA followed by Tukey’s test. Values are means ± SD of three independent biological replicates. *** P< 0.001, Student’s t-test compared to Col-0, in (A) and (B), and compared to VLN1::GFP and ADF7::GFP in (F) and (H).

Fig 3. ADF7 inhibits VLN1-mediated thick bundle formation in the root epidermal cells from different regions and outgrowing root hairs.

(A) Confocal microscopy images of root epidermal cells visualized by the expression of fABD2::GFP in the elongation/differentiation and transition regions from Col-0, adf7-2, ADF7 OE #14, vln1-2, VLN1 OE #8, adf7 vln1 #1, and adf7 VLN1 OE #1 seedlings. Enlarged views from the red boxes are in the bottom row. Scale bar, 20 μm. (B) Violin plot showing the average and contribution of fluorescence intensity of actin cables in (A). The red line represents the average fluorescence intensity in different genotypes. Histogram depicting skewness and percentage of occupancy of F-actin in (A). (C) Confocal microscopy images of root epidermal cells in the root hair region from the primary root tips of 4-d-old seedlings in (A). Enlarged views from the red boxes are in the bottom row. Scale bar, 25 μm. (D) The parameter fluorescence intensity of actin cables, skewness, and percentage of occupancy of F-actin in (C). (E) Confocal microscopy images of outgrowing root hair cells from the primary root tips of 4-d-old seedlings in Col-0, adf7-2, vln1-2, and adf7 vln1 #1 in cross-section. Enlarged views from the red boxes are in the bottom row. Scale bar, 20 μm. (F) The parameter fluorescence intensity of actin cables, skewness, and percentage of occupancy of F-actin in (E). (G) Confocal microscopy images of outgrowing root hair cells from the primary root tips of 4-d-old seedlings in Col-0, adf7-2, vln1-2, and adf7 vln1 #1 in the longitudinal section. Scale bar, 10 μm. (H) The parameter fluorescence intensity of actin cables, skewness, and percentage of occupancy of F-actin in (G). (I) Time-lapse images of newly emerged root hairs of Col-0, adf7-2, vln1-2, and adf7 vln1 #1 for 0, 5, and 10 min. Scale bar, 10 μm. (J) The parameter fluorescence intensity of actin cables, skewness, and percentage of occupancy of F-actin in (I). At least 60 root epidermal cells from at least 20 individual seedlings are calculated in every genotype. Significant difference (P< 0.05) indicated by different letters among genotypes is determined for each condition by one-way ANOVA, followed by Tukey’s test in (B), (D), (F), (H), and (J).

Fig 4. The F-actin dynamics regulated by ADF7-inhibited-VLN1 on single filament level in epidermal cells of root apices.

(A) Depolymerizing processes in Col-0 and adf7-2 seedlings. Scale bar, 10 μm. (B) Severing processes in Col-0 and adf7-2 seedlings. Scale bar, 10 μm. (C) Bundling processes in Col-0, adf7-2, vln1-2, and adf7 vln1#1 seedlings. Scale bar, 10 μm. (D) The parameters of actin dynamics regulated by ADF7 and VLN1 on single actin filament level in epidermal cells of root spices under normal conditions and Lat A treatments. To analyze the bundling frequency, a 30×30 μm² region was selected, At least 60 root cells from at least 20 individual seedlings are calculated in every genotype. Significant difference (P< 0.05) indicated by different letters among genotypes is determined for each condition by one-way ANOVA followed by Tukey’s test.

Fig 5. The F-actin depolymerization regulated by ADF7-inhibited-VLN1 promotes root hair formation.

(A) Images of root hairs from Col-0, adf7-2, ADF7 OE #14, vln1-2, VLN1 OE #8, and adf7 vln1 #1 under CK and Lat A treatments (0.2 μM). Scale bar, 200 μm. (B) Histogram depicting root hair number in (A). (C) Confocal microscopy images of epidermal cells visualized by the expression of fABD2::GFP in root apices from Col-0, adf7-2, vln1-2, and adf7 vln1 #1 seedlings under Lat A treatments (0.2 μM). Enlarged views from the red boxes are in the bottom row. Scale bar, 25 μm. (D) Violin plot showing the average and contribution of fluorescence intensity of actin cables in (C). The red line represents the average fluorescence intensity in different genotypes. (E) Histogram depicting skewness of F-actin in (C). (F) Histogram depicting the percentage of occupancy of F-actin in (C). Significant difference (P< 0.05) indicated by different letters among genotypes is determined for each condition by one-way ANOVA followed by Tukey’s test in (B), (D), (E), and (F).

Fig 6. The F-actin depolymerization co-regulated by ADF7-inhibited-VLN1 elevates ROS accumulation during root hair formation.

(A) Images of ROS levels in roots of Col-0, adf7-2, vln1-2, and adf7 vln1 #1 seedlings under Lat A treatments (0.2 μM) and CD (3 μM). Seedlings were treated with H2DCF–DA (see Methods), scale bar, 100 μm. (B) Histogram depicting relative fluorescence intensities of ROS accumulation in (A) At least 30 seedlings were examined for each material in different treatments. (C) Images of root hairs from Col-0, adf7-2, vln1-2, ADF7 OE #13, vln1-2, VLN1 OE #7, adf7 vln1 #1, and adf7 VLN1 OE #1 under 50 μM and 75 μM DPI treatments. A negative control was supplemented with 0.75% DMSO. Scale bar, 200 μm. (D) Histogram depicting root hair number in (C). (E) RT-qPCR quantification of RHD2 expression level in Col-0, adf7-2, vln1-2, and adf7 vln1 #1. Values are means ± SD of three independent biological replicates. *** P < 0.001, Student’s t-test compared to Col-0. Significant difference (P< 0.05) indicated by different letters among genotypes is determined for each condition by one-way ANOVA, followed by Tukey’s test in (B) and (D).

Fig 7. The pathway of ADF7-inhbited-VLN1 is crucial for root hair formation in plant osmotic stress tolerance.

(A) Relative expression level of ADF7 and VLN1 in roots from 6-d-old Col-0 seedlings with 200, 250, and 300 mM mannitol treatments. Values are means ± SD of three independent biological replicates. *** P< 0.001, Student’s t-test compared with Col-0. (B) GUS activity of 4-d-old seedlings carrying the pADF7::GUS or pVLN1::GUS reporter gene with 0, 250, and 300 mM mannitol treatments. Scale bar, 50 μm. (C) Western blotting using proteins extracted from different genotypes carrying the pADF7::ADF7-GFP gene or pVLN1::VLN1-GFP gene with 0, 250, and 300 mM mannitol treatments. Rubisco was used as a loading control. Histograms depict quantification of the relative grayscale value. Values are means ± SD of three independent biological replicates. ** P< 0.01, *** P < 0.001, Student’s t-test compared to Col-0 with 0 mM mannitol treatment. (D) Histogram depicting root hair number from ADF7 and VLN1 genotypes under 0, 200, 250, and 300 mM mannitol treatments. (E) Images of growth state from ADF7 and VLN1 genotype seedlings under 0, 250, and 300 mM mannitol treatments. Scale bar, 1 cm. (F) to (H) Phenotypic characteristics of leaf area (F), dry weight (G), water content (H) from 16-d-old genotype seedlings under 0, 250, and 300 mM mannitol treatments. Significant differences (P< 0.05; indicated by different letters) among genotypes are determined for each condition by one-way ANOVA followed by Tukey’s test in (D), (F), (G), and (H).

Fig 8. The pathway of ADF7-inhibited-VLN1 is involved in osmotic-induced F-actin depolymerization and ROS accumulation.

(A) Confocal microscopy images of epidermal cells visualized by the expression of fABD2::GFP in root apices from Col-0 seedlings under mannitol treatments (200 mM) for 0, 3, 6, and 9 h. Scale bar, 20 μm. (B) Violin plot showing the average and contribution of fluorescence intensity of actin cables in different genotypes seedlings under 200 mM mannitol treatments for different times. The red line represents the average fluorescence intensity in different genotypes. (C) Histogram depicting skewness of F-actin in (B). (D) Histogram depicting the percentage of occupancy of F-actin in (B). (E) ROS production in the roots of Col-0 seedlings under mannitol treatments (200 mM) for 0, 3, 6, and 9 h. Scale bar, 100 μm. (F) Histogram depicting relative fluorescence intensities of the ROS levels in (E). (G) Working model of ADF7 and VLN1 in root hair formation and in response to osmotic stress in Arabidopsis. Arrows represent positive regulation, and bar ends mean inhibitory action. Osmotic stress induces ADF7 expression, which leading to the inclined F-actin depolymerization and the inhibited VLN1 expression that decreases F-actin bundle. The cooperation of ADF7 and VLN1 results in the increased F-actin depolymerization, finer F-actin and F-actin turnover, which associated with the increase ROS level and root hair formation in osmotic stress, then enhancing plant osmotic stress tolerance. At least 30 seedlings were examined for each genotype in different treatments. Significant difference (P< 0.05) indicated by different letters among genotypes is determined for each condition by one-way ANOVA, followed by Tukey’s test in (B), (C), (D), and (F).