Fig 1.
Effects of TSA on pollen germination and mean tube length in Picea wilsonii.
Pollen germination and tube elongation were inhibited in a dose-dependent manner. Pollen tubes were cultured in (A) standard medium for 24 h, (B) medium containing 0.1 μM TSA for 24 h, (C) medium containing 0.5 μM TSA for 24 h, (D) medium containing 1 μM TSA for 24 h, (E) medium containing 2 μM TSA for 24 h, and (F) medium containing 4 μM TSA for 24 h. (G) Percent germination of pollen tubes cultured in media containing various TSA concentrations. (H) Mean lengths of pollen tubes cultured in media containing various TSA concentrations. One asterisk indicates a significant difference between the growth rates of the drug-treated and control groups at P<0.05. A double asterisk indicates a significant difference between the growth rates of the drug-treated and control groups at P<0.01. Bar = 200 μm.
Fig 2.
Effects of NaB on Picea wilsonii pollen germination and mean tube length.
(A) Effects of NaB on pollen tube growth. Mean length of pollen tubes treated with 0, 0.05, 0.1, 0.2, 0.5, and 1 mM NaB, respectively. (B) Effects of NaB on pollen tube germination. Percent germination of pollen tubes treated with 0, 0.05, 0.1, 0.2, 0.5, and 1 mM NaB, respectively. (C) Pollen tubes cultured in standard medium for 24 h. (D) Pollen tubes cultured in medium containing 0.05 mM NaB for 24 h. (E) Pollen tubes cultured in medium containing 0.1 mM NaB for 24 h. (F) Pollen tubes cultured in medium containing 0.2 mM NaB for 24 h. (G) Pollen tubes cultured in medium containing 0.5 mM NaB for 24 h. (H) Pollen tubes cultured in medium containing 1 mM NaB for 24 h. One asterisk indicates a significant difference between the growth rates of the drug-treated and control groups at P<0.05. A double asterisk indicates a significant difference between the growth rates of the drug-treated and control groups at P<0.01. Three asterisks indicate a significant difference between the growth rates of the drug-treated and control groups at P<0.001. Bar = 150 μm.
Fig 3.
Effect of inhibitors of HDAC on cytosolic Ca2+ in Picea wilsonii pollen tubes.
(A) Fluo-3/AM staining of pollen tubes cultured in standard medium for 24 h. (B) Fluo-3/AM staining of pollen tubes incubated in standard medium supplemented with 0.2% DMSO for 24 h. (C) The fluorescence intensity was analyzed using ImageJ from the base to the tip of the (A) pollen tube. The slope coefficient became larger. (D) The fluorescence intensity was analyzed using ImageJ from the base to the tip of the (B) pollen tube. The slope coefficient became larger. (E) Fluo-3/AM staining of pollen tubes incubated in medium supplemented with 0.5 μM TSA for 24 h. (F) Fluo-3/AM staining of pollen tubes incubated in medium supplemented with 0.5 mM NaB for 24 h. (G) The fluorescence intensity was analyzed using ImageJ from the base to the tip of the (E) pollen tube. The slope coefficient was largely unchanged. (H) The fluorescence intensity was analyzed using ImageJ from the base to the tip of the (F) pollen tube. The slope coefficient was largely unchanged. Bar = 50 μm.
Fig 4.
Treatment of Picea wilsonii pollen tubes with inhibitors of HDAC induces the redistribution of actin filaments.
(A) Pollen tubes cultured in standard medium for 24 h showing actin filaments parallel to the direction of pollen tube elongation. (B) The addition of 0.2% DMSO to the standard medium followed by incubation for 24 h had no obvious effect on the distribution of the actin filaments compared with the controls. (C) In pollen tubes treated with 0.5 μM TSA-containing medium for 24 h, the actin filaments became obviously twisted and irregularly arranged. (D) In pollen tubes cultured in 0.5 mM NaB-containing medium for 24 h, the actin filaments were broken and disorganized. Bar = 20 μm.
Fig 5.
Time course of FM4-64 uptake in a growing Picea wilsonii pollen tube.
(A–E) FM4-64 uptake into the pollen tube followed a strict time sequence, and dye uptake occurred mainly along the apex in a normally growing pollen tube of Picea wilsonii. (F–H) FM4-64 staining of a 0.2% DMSO-treated pollen tube. FM4-64 internalization occurred in the apical region of a growing pollen tube. (K–O) FM4-64 staining of a 0.5 μM TSA-treated pollen tube, showing nonuniform internalization from both sides of the tube. (P–T) FM4-64 staining of a pollen tube cultured in 0.5 mM NaB-containing medium for 24 h. The fluorescence was distributed unevenly and showed no distinct direction during the course of uptake. Bar = 20 μm (arrows indicate the direction of dye distribution).
Fig 6.
Fluorescent immunolabeling of pectins in a Picea wilsonii pollen tube.
(A) JIM5 labeling of pollen tubes cultured in standard medium. Strong fluorescence was detected along the entire tube shank wall, excluding the tip. (B) JIM7 labeling of pollen tubes cultured in standard medium. Esterified pectins were found at the tip of the pollen tube. (C) JIM5 labeling of pollen tubes treated with medium containing 0.2% DMSO. The result is consistent with that obtained for the control (A). (D) JIM7 labeling of pollen tubes treated with medium containing 0.2% DMSO. The distribution of esterified pectins is consistent with that seen in the control (B). (E) JIM5 labeling of pollen tubes cultured in the presence of 0.5 μM TSA. The fluorescence signal was distributed along the pollen tube wall. (F) JIM7 labeling of pollen tubes incubated in medium containing 0.5 μM TSA. Esterified pectins were found only in the basal part of the pollen tube wall. (G) JIM5 labeling of pollen tubes cultured in the presence of 0.5 mM NaB. (H) JIM7 labeling of pollen tubes incubated in medium containing 0.5 mM NaB. Bar = 50 μm.
Fig 7.
Effect of inhibitors of HDAC on callose deposition in Picea wilsonii pollen tube walls.
(A) Control pollen tubes labeled with aniline blue. The fluorescence was distributed evenly along the tube shank, but was absent in the tip region. (B) Pollen tubes treated with 0.2% DMSO. The fluorescence distribution is consistent with that in the control. (C) Pollen tubes cultured in medium containing 0.5 μM TSA. Strong fluorescence was detected at the tip. (D) Pollen tubes cultured in medium containing 0.5 mM NaB. Bar = 50 μm.