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VAMP726 and VAMP725 regulate vesicle secretion and pollen tube growth in Arabidopsis

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Abstract

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VAMP726/VAMP725 and SYP131 can form a part of a SNARE complex to mediate vesicle secretion at the pollen tube apex.

Abstract

Secretory vesicle fusion with the plasma membrane of the pollen tube tip is a key step in pollen tube growth. Membrane fusion was mediated by SNAREs. However, little is known about the composition and function of the SNARE complex during pollen tube tip growth. In this study, we constructed a double mutant vamp725 vamp726 via CRISPR‒Cas9. Fluorescence labeling combined with microscopic observation, luciferase complementation imaging, co-immunoprecipitation and GST pull-down were applied in the study. We show that double mutation of the R-SNAREs VAMP726 and VAMP725 significantly inhibits pollen tube growth in Arabidopsis and slows vesicle exocytosis at the apex of the pollen tube. GFP-VAMP726 and VAMP725-GFP localize mainly to secretory vesicles and the plasma membrane at the apex of the pollen tube. In addition, fluorescence recovery after photobleaching (FRAP) experiments showed that mCherry-VAMP726 colocalizes with Qa-SNARE SYP131 in the central region of the pollen tube apical plasma membrane. Furthermore, we found that VAMP726 and VAMP725 can interact with the SYP131. Based on these results, we suggest that VAMP726/VAMP725 and SYP131 can form a part of a SNARE complex to mediate vesicle secretion at the pollen tube apex, and vesicle secretion may mainly occur at the central region of the pollen tube apical plasma membrane.

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Acknowledgements

We thank the Arabidopsis Biological Resource Center at Ohio State University for providing the T-DNA insertion lines. We thank Dr. Zhenbiao Yang (University of California, Riverside) for the gift of the plasmid contained AtPRK1; Dr. Qijun Chen (China Agricultural University) for the gift of CRISPR-Cas9 vector; Dr. Yingzhang Li (China Agricultural University) for the gift of the plasmids pCAMBIA1300-nLUC-GUS and pCAMBIA1300-GUS-cLUC.

Funding

This study was supported by the National Natural Science Foundation of China (31270230 and 32270363).

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Authors and Affiliations

  1. State Key Laboratory of Plant Environmental Resilience, College of Biological Sciences, China Agricultural University, Beijing, 100193, China

    Xinyan Liu, Dandan Zhu, Fuli Zhao, Yadan Gao, Jianji Li & Yan Li

Authors
  1. Xinyan Liu
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  2. Dandan Zhu
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  3. Fuli Zhao
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  4. Yadan Gao
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  5. Jianji Li
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  6. Yan Li
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Contributions

YL, XL and DZ conceived and designed the experiments. XL, DZ, FZ, YG, JL and YL performed the experiments. XL, DZ, FZ and YL analyzed the data. XL and YL wrote the paper. All authors contributed to the discussion of the results and edited and approved the submitted version of the manuscript.

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Correspondence to Yan Li.

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The authors declare that they have no conflicts of interest.

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Communicated by Xian Sheng Zhang.

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Supplementary Information

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299_2023_3075_MOESM1_ESM.zip

Supplementary file 1: Fig. S1. Promoter–GUS assay of VAMP726 expression in Arabidopsis. GUS histochemical activity assay showed that VAMP726 is abundantly expressed in Arabidopsis pollen and pollen tubes. A Seedling. B Inflorescence. C Flower. D Pistil. E Pollen. F Pollen tube. Fig. S2. Identification of the vamp726 mutant. A Schematic view of the genomic structure of VAMP726, and sites of the vamp726 T-DNA insertion. The solid boxes represent exons and the intervening lines represent introns. The location and orientation of T-DNA insertion are indicated. B Identification of T-DNA insertion homozygous of VAMP726 by PCR. C RT-PCR analysis of the vamp726 homozygous mutant line, indicated that there was no VAMP726 transcripts in the mutant. Actin8 was amplified as the positive internal control. D There was no significant difference in the length of pollen tubes of WT and vamp726 grown in vivo for 8 h, and the white arrow indicated the position of pollen tube growth. Bar = 500 μm. E Statistical analysis of the pollen tube relative length in vivo. To reduce the impact of individual differences, we calculated the relative length of the pollen tubes to the female tissues. 7 < n < 15, mean ± SD, P > 0.05 (Student’s t-test). At least 3 independent replicates were performed with consistent results. F WT and vamp726 pollen tubes grew in vitro for 5 h, n > 300, P > 0.05 (Student’s t-test). At least 3 independent replicates were performed with consistent results. Fig. S3. Identification of the vamp725 mutant. A CRISPR/Cas9-mediated gene editing knocked out VAMP725 in background of WT to generate a mutant vamp725. Sequence analysis showed 6-base deletion of AGAATT and a subsequent insertion of a base a occur at that target site, resulting in a frame shift and a stop codon at the 20th amino acid after the target site. B There was no significant difference in the length of pollen tubes of WT and vamp725 grown in vivo for 8 h, and the white arrow indicated the position of pollen tube growth. Bar = 500 μm. C Statistical analysis of the pollen tube relative length in vivo. To reduce the impact of individual differences, we calculated the relative length of the pollen tubes to the female tissues. 7 < n < 15, P > 0.05 (Student’s t-test). At least 3 independent replicates were performed with consistent results. D WT and vamp725 pollen tubes grew in vitro for 5 h, n > 300, P > 0.05 (Student’s t-test). At least 3 independent replicates were performed with consistent results. Fig. S4. Identification of the vamp725 vamp726 double mutant. A The sequence similarity of VAMP726 and VAMP725 is 69.82%. B CRISPR/Cas9-mediated gene editing knocked out VAMP725 in background of vamp726 to generate a double mutant vamp725 vamp726. Sequence analysis showed four bases AATT deletion at the target sequence. C RT-PCR analysis of the vamp725 vamp726 homozygous mutant line indicated that there was no VAMP726 transcripts in the double mutant. In the two homozygous lines which was complemented with GFP-VAMP726 driven by LAT52 could detect the expression of VAMP726. Actin8 was amplified as the positive internal control. D The pollen germination rates for 1 h, 2 h, 3 h, 4 h and 5 h respectively. This image showed that there was no significant difference in the germination rate between the WT and double mutant in vitro. n=5, P>0.05 (Student’s t-test). Fig. S5. Alexander staining for the mature pollen of vamp725 vamp726 mutant and WT. The pollen of WT and vamp725 vamp726 mutant were stained with Alexander, and they were uniformly red, indicating normal activity of the mutant pollen grains. Bar= 20 μm. Fig. S6. DAPI staining for the mature pollen of vamp725 vamp726 mutant and WT. DAPI staining showed that the mutant had normal karyotype during pollen development. These results indicated that pollen development was not affected obviously in the mutant. Bar= 20 μm. Fig. S7. The statistical analysis for Alexander and DAPI staining. (50 < n < 140), mean±SD, P > 0.05 (Student’s t-test). Fig. S8. The statistical analysis for the seed sets of the vamp725 vamp726 mutant and WT. (n=30), mean±SD, P > 0.05 (Student’s t-test). The average seed numbers of a mature silique in the vamp725 vamp726 mutant were 53.53 ±4.54. The average seed numbers of a mature silique in WT were 53.8 ±3.51. These results indicated that the seed sets was not obviously affected in the mutant. Fig. S9. VAMP725-GFP could rescue the phenotype of vamp725 vamp726 mutant. A The pollen tube growth patterns of WT, vamp725 vamp726 and two complemented lines which expressed VAMP725-GFP driven by LAT52 in vivo for 8 h. The pollen tube length of the mutant was short than that of WT and complemented lines. The result showed that VAMP725-GFP could rescue the phenotype of vamp725 vamp726. Bar = 500 μm. B Statistic analysis for the relative lengths of the pollen tube growth in vivo for 8 h. 10<n<15, **P < 0.01, Student’s t-test. C The subcellular localization of VAMP725-GFP in the pollen tubes of Arabidopsis. Top was the subcellular localization of VAMP725-GFP in WT and down was in vamp725 vamp726. VAMP725-GFP mainly localized on the inverted-cone-shaped region in the pollen tube tip and some also resided on the pollen tube plasma membrane. Bar= 5 μm. D Colocalization of VAMP725-GFP and FM4-64 in the pollen tube of Arabidopsis. The fluorescence distributions of VAMP725-GFP and FM4-64 staining were largely colocalization in the pollen tube tip. Bar = 10 μm. Fig. S10. The subcellular localization of GFP-VAMP726 in tobacco pollen tube. GFP-VAMP726 which was transformed through bombardment transformation mainly located on the inverted-cone-shaped region in the tobacco pollen tube tip. GFP-VAMP726 also resided on the apical plasma membrane of the tobacco pollen tube. Bar = 10 μm. Fig. S11. The localization of GFP-VAMP726 or mCherry-VAMP726 in lower expressing level in the pollen tubes. A The subcellular localization of GFP-VAMP726 driven by a native promoter of VAMP726 in the pollen tube of Arabidopsis. GFP-VAMP726 localized at the pollen tube tip and the apical plasma membrane. Bar=10 μm. B The subcellular localization of mCherry-VAMP726 in lower expressing level in the tobacco pollen tube. mCherry-VAMP726 also localized in the apical plasma membrane and pollen tube tip. Bar=5 μm. Fig. S12. Brefeldin A treatments resulted in GFP-VAMP726 to diffuse in pollen tube tip of tobacco. The growing tobacco pollen tube expressing GFP-VAMP726 was treated with 14 μM BFA, and observed by using a confocal microscope. After Brefeldin A treatments, the inverted-cone of GFP-VAMP726 in the pollen tube tip was gradually disappeared. The localization of GFP-VAMP726 was normal with pollen tube growth when methanol was used as the control. Bar = 10 μm. Fig. S13. VAMP726 and SYP131 colocalized at the apical plasma membrane in the tobacco pollen tube. A mCherry-VAMP726 mainly localized on the inverted-cone-shaped region and apical plasma membrane of the tobacco pollen tube. The SYP131-GFP wasresided on the whole plasma membrane of the tobacco pollen tube. Merged the two channels showed that mCherry-VAMP726 and SYP131-GFP colocalized at the apical plasma membrane of the pollen tube. Bar = 10 μm. B Quantitative analysis for the fluorescence intensity of mCherry-VAMP726 and SYP131-GFP along the white line in the pollen tube of tobacco. The image showed that both mCherry-VAMP726 and SYP131-GFP had a sharp fluorescence intensity increase on the apical plasma membrane of the pollen tube. The data indicated that mCherry-VAMP726 and SYP131-GFP colocalized at the apical plasma membrane of the tobacco pollen tube. Fig. S14. VAMP725 can interact with SYP131. A Firefly luciferase complementation assay showed that VAMP725 interacted with SYP131 in the tobacco leaf. nLUC+cLUC-SYP131 and nLUC +cLUC-VAMP725 were transformed as negative controls. SGT1-Nluc+Cluc-RAR1 were transformed as a positive control. The interaction between cLUC-VAMP725 and nLUC-SYP131 had an obvious fluorescent signal while there was no fluorescent signal in the negative controls. At least 10 tobacco leaves were collected and analyzed, which had similar results. B Co-IP assays showed the interaction between VAMP725 and SYP131. Protein extracted from the tobacco leaf which expressed Flag-SYP131+Myc-VAMP725 as the experimental group, and Flag+Myc-VAMP725 as control were subjected to immunoprecipitation with anti-Flag beads. Then Western blotting was performed with the anti-Myc and anti-Flag monoclonal antibodies. The results indicated that Flag-SYP131 could bind with Myc-VAMP725, no Myc-VAMP725 band was detected in the control group. The Flag-tag was too small and run out in the SDS-PAGE, so the Flag band was not shown in the results. The full-length blot is presented in Fig. S15. The experiments were repeated for three times with similar results. Fig. S15. The original immunoblotting images. A The original immunoblotting image of Figure 7C (Input, anti-Flag). B The original immunoblotting image of Figure 7C (Input, anti-Myc). C The original immunoblotting image of Figure 7C (IP, anti-Flag). D The original immunoblotting image of Figure 7C (IP, anti-Myc). E The original immunoblotting image of Figure 7D (Input and pull-down, anti-His). F The original immunoblotting image of Figure 7D (Input and pull-down, anti-GST). G The original immunoblotting image of Figure S14B (Input, anti-Flag). H The original immunoblotting image of Figure S14B (Input, anti-Myc). I The original immunoblotting image of Figure S14B (IP, anti-Flag). J The original immunoblotting image of Figure S14B (IP, anti-Myc).

Supplementary file 2: Video 1. FRAP of AtPRK1-GFP in the apical plasma membrane of Arabidopsis pollen tube.

Supplementary file 3: Video 2. FRAP of AtPRK1-GFP in the apical plasma membrane of vamp725 vamp726 pollen tube.

Supplementary file 4: Video 3. GFP-VAMP726 in the Arabidopsis growing pollen tube.

Supplementary file 5: Video 4. Brefeldin A treatments resulted in mCherry-VAMP726 to diffuse in the Arabidopsis pollen tube tip.

Supplementary file 6: Video 5. The localization of mCherry-VAMP726 was normal with pollen tube growth when methanol was used as the control.

Supplementary file 7: Video 6. Brefeldin A treatments resulted in GFP-VAMP726 to diffuse in the tobacco pollen tube tip.

Supplementary file 8: Video 7. The localization of GFP-VAMP726 in the tobacco pollen tube tip was normal with rapid pollen tube growth when methanol was used as the control.

Supplementary file 9: Video 8. FRAP assay showed that mCherry-VAMP726 first recovered at the central region of apical plasma membrane in the pollen tube.

Supplementary file 10: Table S1. Primer sequences were used in the manuscript.

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Liu, X., Zhu, D., Zhao, F. et al. VAMP726 and VAMP725 regulate vesicle secretion and pollen tube growth in Arabidopsis. Plant Cell Rep 42, 1951–1965 (2023). https://doi.org/10.1007/s00299-023-03075-w

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  • DOI: https://doi.org/10.1007/s00299-023-03075-w

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