VPS18-regulated vesicle trafficking controls the secretion of pectin and its modifying enzyme during pollen tube growth in Arabidopsis

Abstract

In eukaryotes, homotypic fusion and vacuolar protein sorting (HOPS) as well as class C core vacuole/endosome tethering (CORVET) are evolutionarily conserved membrane tethering complexes that play important roles in lysosomal/vacuolar trafficking. Whether HOPS and CORVET control endomembrane trafficking in pollen tubes, the fastest growing plant cells, remains largely elusive. In this study, we demonstrate that the four core components shared by the two complexes, Vacuole protein sorting 11 (VPS11), VPS16, VPS33, and VPS18, are all essential for pollen tube growth in Arabidopsis thaliana and thus for plant reproduction success. We used VPS18 as a representative core component of the complexes to show that the protein is localized to both multivesicular bodies (MVBs) and the tonoplast in a growing pollen tube. Mutant vps18 pollen tubes grew more slowly in vivo, resulting in a significant reduction in male transmission efficiency. Additional studies revealed that membrane fusion from MVBs to vacuoles is severely compromised in vps18 pollen tubes, corroborating the function of VPS18 in late endocytic trafficking. Furthermore, vps18 pollen tubes produce excessive exocytic vesicles at the apical zone and excessive amounts of pectin and pectin methylesterases in the cell wall. In conclusion, this study establishes an additional conserved role of HOPS/CORVET in homotypic membrane fusion during vacuole biogenesis in pollen tubes and reveals a feedback regulation of HOPS/CORVET in the secretion of cell wall modification enzymes of rapidly growing plant cells.

Figure 1

vps18 shows retarded pollen tube growth in vivo. A, Aniline blue staining of wild-type pistils showing pollen tube growth at 6 HAP after they were pollinated with sufficient numbers of either complementing pollen grains that contain VPS18–GFP fusion protein in the vps18 mutant background (n >90) or vps18 pollen grains (n >90) as indicated. B, Statistical results of the mean length of complemented and vps18 pollen tubes in vivo at 6 HAP. Error bars represent standard deviation of the mean. ***P ˂ 0.01. n = 9. C, Aniline blue staining of wild-type pistils at 10 HAP after pollination as in (A). D, Statistical results of the mean length of complemented and vps18 pollen tubes in vivo at 10 HAP. Error bars represent standard deviation of the mean. ***P ˂ 0.01. n = 9. The vertical white lines in (A) and (C) indicate the position that the majority of pollen tube tips have reached. E, Aniline blue staining of wild-type pistils at 48 HAP after pollination as in (A). Asterisks indicate unfertilized ovules, whereas arrowheads represent fertilized ovules. Student’s t tests were used to analyze the statistical significance of the results presented in B and D. vps18^–/GFP⁺ is an abbreviation for vps18^–/VPS18_pro:VPS18-LAT52:GFP+. Scale bars: 200 μm.

Figure 2

Expression pattern of VPS18 in A. thaliana and subcellular localization of the encoded protein in pollen tubes. A, GUS staining patterns of VPS18 in inflorescences, B, in mature anthers and siliques, C, in pollen grains and pollen tubes as well as D, in young seedlings. E–G, Localization of VPS18-GFP and 2S albumin-mRFP in pollen tubes as indicated at 4 HAG. H, Pearson’s coefficient of the co-localization pattern shown in (G). Error bars represent standard deviation of the mean. n = 10. I–K, Localization of VPS18–GFP fusion protein and VAMP711–mCherry fusion protein in pollen tubes as indicated at 4 HAG. L, Pearson’s coefficient of the co-localization pattern shown in (K). Error bars represent standard deviation of the mean. n = 7. Gene constructs are indicated. Scale bars: 500 μm (A, B, D), 20 μm (C), and 10 μm (E, F, G, I, J, and K).

Figure 3

MVB-to-vacuole trafficking is impaired in vps18 pollen tubes. A, B, Localization of 2S albumin-mRFP. C, D, VAMP711-mCherry in pollen tubes at 6 HAG in the wild-type (WT) and mutant vps18 pollen tubes, respectively. E–H, Internalization and transport of FM4-64 in the wild-type and vps18 pollen tubes. Pollen tubes were stained at 2 HAG. Uptake of FM4–64 in mutant pollen tubes complemented with VPS18–GFP (E) and vps18 pollen tubes (F) at 10 min after dye treatment. Transport of FM4-64 in complemented (G) and vps18 pollen tubes (H) at 240 min after dye treatment. (Left images in A–H) Magenta, (right images in A–D) bright field, and (right images in E–H) green channel, respectively. The dashed lines in (F) and (H) indicate the contours of vps18 pollen tubes. Note that vps18^–/GFP⁺ is the abbreviation for vps18^–/VPS18_pro:VPS18-LAT52:GFP⁺. Scale bars: 10 μm (A–D) and 5 μm (E–H).

Figure 4

Secretory events are increased in vps18 pollen tube tips. A–D, TEM images of the wild-type (vps18 complemented with VPS18–GFP) and vps18 mutant pollen tubes are shown at 4 HAG. Shank regions are visible in (A, B) and the tip region in (C, D). Black rectangles indicate tip areas that are enlarged. Asterisks mark MVB-like vesicles and arrows point towards extracellular vesicles. V, vacuole; SV, secretory vesicle. E–G, Dynamics of mRuby3-RabA4d-labeled vesicles in the wild-type as above and mutant pollen tube tips in a time series as indicated at 4 HAG. Numbers at the bottom display time points elapsed from the beginning of the experiment. G, Quantitative analysis of the oscillation of fluorescence intensity at the tip of growing wild-type (n = 8) and vps18 mutant pollen tubes (n = 10). Mean intensity of fluorescence was measured in the area marked by the white circle (shown in E). Confocal images were collected every 5 s for a total duration of 5 min. H, I, Localization of mRuby3-RabA4d in the wild-type (H) and vps18 (I) pollen tubes at 20 min after incubation with BFA. J, Red fluorescence pixel intensity is measured along the pollen tube from the apex. n >30. K, L, Localization of mRuby3-RabA4d in the wild-type (K) and vps18 (L) pollen tubes at 60 min after incubation with BFA. M, Red fluorescence pixel intensity is measured along the pollen tube from the apex. n >30. N, O, Localization of mRuby3-RabA4d in the wild-type (N) and vps18 (O) pollen tubes at 120 min after incubation with BFA. P, Magenta fluorescence pixel intensity was measured along the pollen tube from the apex. n >30. E, F, Merged magenta and green channel, (upper images in H, I, K, L, N, and O) magenta and (lower images in H, I, K, L, N, and O) green channel, respectively. The dashed lines in I, L, and O indicate the contours of vps18 pollen tubes. Note that vps18^–/GFP⁺ is the abbreviation for vps18^–/VPS18_pro:VPS18-LAT52:GFP⁺. Scale bars: 1 μm (A–D), 100 nm in the enlargements shown in black rectangles and 10 μm (E, F, H, I, K, L, N, and O).

Figure 5

Deposition of pectin and PMEs is enhanced in vps18 pollen tubes. A–F, Distribution and deposition of methyl-esterified pectin and de-esterified pectin in pollen tubes at 4 HAG. Methyl-esterified pectin in complemented (A) and vps18 pollen tubes (B) is labeled by the JIM7 antibody and de-esterified pectin accordingly (D, E) by the LM19 antibody. C, F, Fluorescence pixel intensity at the cell wall measured along with the JIM7/LM19 signals from the apex. n >30. G–L, Localization of PMEs in pollen tubes. Localization and fluorescence intensity of VGD1-mRuby3 in complemented (G) and vps18 pollen tubes (H) at 4 HAG. Localization and fluorescence intensity of PPME1-mRuby3 in complemented (J) and vps18 pollen tubes (K) at 4 HAG. I, L, Fluorescence pixel intensity at the cell wall measured along the pollen tube from the apex. n >30. M, Relative transcript levels of VPS18 in pollen tubes from vps18^–/–/VPS18_pro:VPS18-LAT52:GFP^+/+ (WT) plants and vps18^–/^–/VPS18_pro:VPS18-LAT52:GFP^+/– (vps18^+/–) plants. N, Relative transcript levels of VGD1 and PPME1 in pollen tubes from vps18^–/–/VPS18_pro:VPS18-LAT52:GFP^+/+ (WT) plants and vps18^–/–/VPS18_pro:VPS18-LAT52:GFP^+/– (vps18^+/–) plants. O, A proposed model of coordinated regulation between MVB-to-vacuole trafficking and secretory events. In vps18 pollen tubes, fusion from MVBs to vacuoles is disrupted and results in excessive accumulation of MVB-like vesicles. As a consequence of feedback regulation, more exocytic vesicles are released from the TGN, which results in excessive secretion of pectin and PMEs to the apical cell wall. Increased deposition of pectin inhibits the fast growth of pollen tubes. A, B, D, E, Merged magenta and green channel, (upper images in G–K) red, and (lower images in G–K) green channel, respectively. The dashed lines in H and K indicate the contours of vps18 pollen tubes. Error bars represent standard deviation of the mean. Student’s t tests were used to analyze the statistical significance of the results. **P ˂ 0.05. ***P ˂ 0.01. Note that vps18^–/GFP⁺ is the abbreviation for vps18^–/VPS18_pro:VPS18-LAT52:GFP⁺. Scale bars: 10 μm.