Figure 1.
Pollen germination and pollen tube growth were inhibit in RNAi transgenic tobacco.
A large population of pollen tubes of RNAi tobacco(A) compared with wild-type pollen tubes(B) to illustrate the difference of both length and number of pollen tubes; C: Western-blotting with anti-NtGNL1 to confirm the RNAi effects on pollen. NtGNL1 expression in RNAi lines. Anti-actin was used as an endogenous standard; wild-type SR1 was used as control. Bar = 100 µm.
Figure 2.
Inhibiting NtGNL1 disturbed the temporal sequence of FM4-64 uptake in pollen tube.
A–E: controls (wild-type pollen tubes) showing a strict time sequence of FM4-64 uptake with the fluorescence distributed evenly at the apex. F–J: RNAi transgenic pollen tubes. Stars indicate laggard vesicles. Pictures on the left lane are bright field images of corresponding pollen tubes. Pollen tubes were cultured for 3 h. Bar = 10 µm.
Figure 3.
Distribution patterns of endosomes in RNAi pollen tubes were altered after down-regulating NtGNL1 Expression.
A and D are bright field images; others are fluorescent images or schematic diagrams. A–C: Control, showing normal fluorescence distribution in a converse V-shape. D–F: RNAi pollen tube showing fluorescence accumulated at the sub-apical region. The arrows indicate the direction of the vesicle movement. Images were taken between 30 and 60 min after loading FM4-64 to 3 h-cultured pollen tubes. Bar = 10 µm.
Figure 4.
Ultrastructural observation of abnormal post-Golgi trafficking in RNAi pollen tubes.
A, C, E: control (wild-type pollen). B, D, F: RNAi pollen tubes. A, B: Tip region of pollen tubes shows more small vesicles at the tip region I in control than in RNAi transgenic pollen tubes. Bar = 3 µm. C, D: more bigger vesicles at region II in RNAi transgenic than in control. Bar = 400 µm. E, F: Showing the volume of putative MVB in wild-type (E) and RNAi pollen tubes (F). Notice the comparative diameter of the putative PVCs/MVBs and adjacent Golgi bodies. Bar = 50 nm in (E); Bar = 100 nm in (F). cw: cell wall; g: Golgi bodies. M: putative PVCs/MVBs; V: vesicle.
Table 1.
Various diameter of vesicles (%) compare in region II.
Figure 5.
Various abnormal structures of Golgi apparatus in NtGNL1 RNAi lines.
A: Wide type pollen tube showing separated Golgi bodies. B: Accumulated Golgi bodies in RNAi pollen tubes. C: Segmentation of Golgi bodies. D: Wild type Golgi body. E: Normal Golgi apparatus in RNAi plants. F: Cisternal Golgi curved into a ring. G: Expanded and fragmented Golgi stacks. H–J: ER-Golgi hybrids. K: Normal ER in RNAi pollen tubes. Bar = 400 µm. ER: Endoplasmic reticulum; g: Golgi stacks. Bar = 200 µm.
Table 2.
Different Golgi bodies ratio (%) in RANi pollen tubes.
Figure 6.
NtGNL1 colocalized with Golgi bodies and PVCs respectively.
A, B: NtGNL1-GFP was transiently expressed in pollen tube. C: Transient expression of ST-DsRED. D: Transient expression of Ntrab5-DsRED. E: Merged image of A and C showing NtGNL1 and Golgi bodies partial colocalization. F: Merged image of B and D, showing NtGNL1 overlapped with PVCs. Bar = 20 µm.
Figure 7.
F-actin disorganization in NtGNL1 RNAi pollen tubes.
A and H: wild type pollen tubes. Others are RANi pollen tubes. A: Wild-type pollen tubes show no straight F-actin bundles at the clear zone. B: Twisted bundles extend to the clear zone of pollen tip(arrow). C: Condensed F-actin bundles (lower arrow) abundantly extend to the tip region of pollen tube (upper arrow). D: Thick bundles accumulate in expanded pollen tube tip and extend perpendicular to the tube long axis. E: Disorganized actin bundles accumulated at the pollen tip. F: Thick and twisted actin bundles bending in RNAi pollen tube. G: Actin fine fringe perpendicular to the plasma membrane (arrow) and absent at the clear zone in control. H: actin fringes collapsed at the sub-region and shifted to the clear zone (arrow). A- F. revealed by LAT52-ABD2-GFP; G and H were labeled by FITC–phalloidin. Bar = 20 µm in A- F. Bar = 10 µm in G and H.