Figure 1.
Effects of DCB on pollen germination and pollen tube growth of Pinus bungeana Zucc.
Bars = 60 µm (A, B, C), 20 µm (F, G). A. The control pollen tube growing in 1% ethanol, showing normal length and shape. B. Pollen tubes cultured in normal medium for 3 d, showing normal shape and long tube. C. Pollen tubes treated with 10−1 µM DCB for 3 d, the tip and /or the base of pollen tube swelled. D. DCB slightly affected germination percentage of pollen grains of Pinus bungeana. Only pollen tubes that were longer than the diameter of pollen grain were measured. Mean values are shown with SD of three experiments. E. DCB inhibited the pollen tube growth of Pinus bungeana. Only pollen tubes that were longer than the diameter of pollen grain were measured. Mean values are shown with SD of three experiments. F. Pollen tubes cultured in normal medium for 3 d, showing normal shape. G. Pollen tubes cultured in normal medium for 3 d and then treated with DCB for 6 h, showing the tube tip ruptured (arrow).
Figure 2.
Effects of DCB on the reorganization of the cytoskeleton in pollen tubes.
Bar = 100 µm. A. Control pollen tubes, showing long pollen tube and normal organization of actin filaments distributed throughout the tube. B. Pollen tubes treated with 10−1 µM DCB, showing actin filaments at the basal part of the tube were destroyed, while the organization of actin filaments at the tip was not affected. C. Pollen tubes treated with 1 µM DCB for 3 d, showing the swollen pollen tubes and the reorganization of actin filaments. D. Pollen tubes treated with 1 µM DCB for 4 d, showing actin filaments were reorganized and mainly vertical to the direction of elongation of the pollen tube. E. In the control pollen tube, microtubules were distributed throughout the pollen tube in a net axial array, mainly parallel to the direction of elongation. F. Pollen tubes treated with 10−1 µM DCB for 4 d, microtubule arrangement was disrupted.
Figure 3.
Effects of DCB on the FM4-64-uptake time course in growing Pinus bungeana pollen tubes.
Bar = 25 µm. A. Confocal fluorescence images of FM4-64 internalization in the control pollen tubes at different times, the rapid uptake suggests a high rate of endocytosis and membrane trafficking in the pollen tube. B. In the DCB-treated pollen tubes, the uptake of FM4-64 occurred at the basal part of tube and the internalization process was activated by DCB treatment.
Figure 4.
Effects of DCB on the ultrastructure of pollen tube of Pinus bungeana Zucc.
Bars = 1 µm (A, C, E), 0.2 µm (B, D, F). CW, cell wall; G, Golgi; M, mitochondria; V, vacuole. A. Tip region of a control normal pollen tube, showing the vesicles and organelles at the apical clear zone. The cell wall was uniform and 0.5 µm in thickness. B. Normal pollen tube tip, showing typical morphology of Golgi and endoplamic reticulum. Golgi contains 6 cisterna and shows a distinct cis-to-trans polarity. C. Pollen tubes treated with 10−1 µM DCB for 3 d, showing the loosen tube wall and the vacuoles at the tip. D. Pollen tubes treated with 10−1 µM DCB for 4 d, the cisternae of Golgi and the membrane of mitochondria began to disrupt (arrow). E. Pollen tubes treated with 1 µM DCB for 3 d, showing the thin tube wall and numerous vacuoles in the pollen tube tip. The cell wall became thinner compared to the control and was about 0.3 µm in thickness. F. Pollen tubes treated with 1 µM DCB for 3 d, showing the disruption of Golgi near the vacuoles.
Figure 5.
Effects of DCB on the distribution of cellulose and callose in tube walls of Pinus bungeana Zucc.
Bars = 20 µm A. Calcofluor stained control pollen tubes, showing cellulose was distributed along the whole length of pollen tube. B. Calcofluor stained DCB-treated pollen tubes, showing less cellulose was present at the pollen tube wall, especially the tube tip. C. Pollen tubes cultured in normal medium for 3 d, showing strong fluorescence, excited from callose on the whole surface of the pollen tube. D. Pollen tubes treated with DCB for 3 d, showing strong fluorescence at the pollen tube tip.
Table 1.
The effects of DCB on cellulose content in the pollen tube of Pinus bungeana Zucc.
Figure 6.
Fluorescence after immunolabeling of Pinus bungeana Zucc. pollen tubes with JIM5 and JIM7.
Bars = 20 µm A. Fluorescence after antibody JIM5 labeling of pollen tubes cultured for 3 d in normal medium (indicated by arrows). B. Corresponding bright-field image showing strong fluorescence occurred at the germination site and the basal part of pollen tube wall. C. Fluorescence was observed mainly at the tube tip after antibody JIM5 labeling of pollen tubes cultured for 3 d in the presence of 10−1 µM DCB (indicated by arrows), D. Corresponding bright-field image showing that faint fluorescence occurred at the germination site and pollen tube tip. E. Fluorescence after antibody JIM7 labeling of pollen tubes cultured for 3 d in normal medium (indicated by arrows). F. Corresponding bright-field image showing that fluorescence occurred at the pollen tube tip. G. Fluorescence after antibody JIM7 labeling of pollen tubes cultured for 3 d in the presence of 10−1 µM DCB (indicated by arrows). H. Corresponding bright-field image showing that fluorescence occurred at the pollen tube tip.
Figure 7.
Fourier transform infrared (FTIR) spectrum obtained from the apical region of Pinus bungeana pollen tubes cultured for 3 d.
A. The FTIR spectra obtained from the tip region of the control pollen tubes and that of treated with different concentrations of DCB revealed that DCB induced changes in absorbance and /or the displacements of the peaks. CK: the control pollen tube. B. The difference spectra generated by digital subtraction of the spectra of control tube walls from the spectra of DCB-treated tube walls, showing that the content of proteins and esterified pectins increased, while the cellulose content decreased.