The observation of each stages during FL-ODNs uptake. doi:10.1371/journal.pone.0059112.gThe pollen tube provides an excellent example of polarized cell growth with rapid extension and the processes of vesicle trafficking visible at the tip [32]. Living pollen tubes are convenient for observing endocytosis with FM4-64, 
a lipophilic probe that fluoresces on binding the plasma membrane [32,33]. Thus, the in vitro growth system of the pollen tube might facilitate research on both A-ODN application in plants and on the molecular mechanism(s) of A-ODN uptake.Here, we used A-ODN inhibition techniques to down-regulate NtGNL1 expression in pollen tubes. Our results revealed that AODN passes through the pollen tube wall in culture JI-101 web medium and works to suppress NtGNL1 expression. A-ODN inhibition resulted in similar phenotypes to those observed in RNAi transgenic plants, indicating the A-ODN worked specifically on its intended target. Thus, we established an alternative and convenient experimental system for gene function analysis in pollen tubes, and theAntisense ODN Inhibition in Pollen TubesFigure 2. The effect of A-ODNs on pollen tube growth and NtGNL1 expression level. A: Inhibition effects among antisense ODNs (1.0 mM). n = 300610. Pollen tubes under ON4 and ON6 treatment were obvious shorter than that under other treatment. Asterisks indicate a significant difference (P,0.05). These data were calculated and analyzed by SPSS (16.0) Independent-Sample T Test. Error bars in the columns represent SD. B: No significant inhibition effect on pollen tubes growth was observed during treatment by sense and random ODNs. n = 300610. C: the effect of antisense ODN treatment on NtGNL1 mRNA expression. D: the comparison of cytotoxic effect between control (sense 1531364 or nonsense) and antisense ON4. All of them displayed around 80 viability. n = 300610. doi:10.1371/journal.pone.0059112.gtechnique may facilitate investigations on the molecular mechanism(s) underlying pollen tube growth.Results A-ODNs Effectively Permeate into Pollen TubesUnlike animal and plant mesophyll cells, pollen tubes typically have thick cell walls, consisting of esterified homogalacturonan (a major pectin component) at the pollen tube tip, and cellulose and callus in the rigid wall behind the tip [34,35]. We first tested whether A-ODNs could pass through the pollen tube wall and plasma membrane by labeling a batch of ODNs with Alexa Fluor 488 to monitor the delivery process. Tracing observations revealed that intense Alexa Fluor 488 fluorescence was detectable PHCCC web within pollen tubes after approximately 1 h of incubation (Fig. 1A.a). The Table 1. Sequences and selected positions of antisense 18204824 ODN.Name ON1 ON2 ON3 ON4 ON5 ON6 ONPosition 196 226 345 883 820 998Sequence(5′?’) GCTGATTAAGGCACCCCA CCCTTGGGCTCTGAAATT CGAAATCCCCACCTCACA CTGGGCCAGCGCACACTT CATGCATCGTGTGGCGTG TCCCCTACGCTCACCAAA CGCTTCAAGCACCCTCTGfluorescently labeled ODN (FL-ODN) first appeared as small dots or patches in the cytoplasm of the pollen tube (Fig. 1A.a), which then accumulated in the tip region (Fig. 1A.b). After 3 h, the signals had dispersed evenly throughout the pollen tube (Fig. 1A.c). During a 2-h co-culture with FL-ODN, most pollen tubes showed a similar distribution pattern of fluorescent signal (Fig. S1). These results indicate that the ODNs could effectively enter the pollen tubes within a short period. To determine whether ODN uptake into pollen tubes occurs via endocytosis, we used FM4-64 to track endosome move.The observation of each stages during FL-ODNs uptake. doi:10.1371/journal.pone.0059112.gThe pollen tube provides an excellent example of polarized cell growth with rapid extension and the processes of vesicle trafficking visible at the tip [32]. Living pollen tubes are convenient for observing endocytosis with FM4-64, a lipophilic probe that fluoresces on binding the plasma membrane [32,33]. Thus, the in vitro growth system of the pollen tube might facilitate research on both A-ODN application in plants and on the molecular mechanism(s) of A-ODN uptake.Here, we used A-ODN inhibition techniques to down-regulate NtGNL1 expression in pollen tubes. Our results revealed that AODN passes through the pollen tube wall in culture medium and works to suppress NtGNL1 expression. A-ODN inhibition resulted in similar phenotypes to those observed in RNAi transgenic plants, indicating the A-ODN worked specifically on its intended target. Thus, we established an alternative and convenient experimental system for gene function analysis in pollen tubes, and theAntisense ODN Inhibition in Pollen TubesFigure 2. The effect of A-ODNs on pollen tube growth and NtGNL1 expression level. A: Inhibition effects among antisense ODNs (1.0 mM). n = 300610. Pollen tubes under ON4 and ON6 treatment were obvious shorter than that under other treatment. Asterisks indicate a significant difference (P,0.05). These data were calculated and analyzed by SPSS (16.0) Independent-Sample T Test. Error bars in the columns represent SD. B: No significant inhibition effect on pollen tubes growth was observed during treatment by sense and random ODNs. n = 300610. C: the effect of antisense ODN treatment on NtGNL1 mRNA expression. D: the comparison of cytotoxic effect between control (sense 1531364 or nonsense) and antisense ON4. All of them displayed around 80 viability. n = 300610. doi:10.1371/journal.pone.0059112.gtechnique may facilitate investigations on the molecular mechanism(s) underlying pollen tube growth.Results A-ODNs Effectively Permeate into Pollen TubesUnlike animal and plant mesophyll cells, pollen tubes typically have thick cell walls, consisting of esterified homogalacturonan (a major pectin component) at the pollen tube tip, and cellulose and callus in the rigid wall behind the tip [34,35]. We first tested whether A-ODNs could pass through the pollen tube wall and plasma membrane by labeling a batch of ODNs with Alexa Fluor 488 to monitor the delivery process. Tracing observations revealed that intense Alexa Fluor 488 fluorescence was detectable within pollen tubes after approximately 1 h of incubation (Fig. 1A.a). The Table 1. Sequences and selected positions of antisense 18204824 ODN.Name ON1 ON2 ON3 ON4 
ON5 ON6 ONPosition 196 226 345 883 820 998Sequence(5′?’) GCTGATTAAGGCACCCCA CCCTTGGGCTCTGAAATT CGAAATCCCCACCTCACA CTGGGCCAGCGCACACTT CATGCATCGTGTGGCGTG TCCCCTACGCTCACCAAA CGCTTCAAGCACCCTCTGfluorescently labeled ODN (FL-ODN) first appeared as small dots or patches in the cytoplasm of the pollen tube (Fig. 1A.a), which then accumulated in the tip region (Fig. 1A.b). After 3 h, the signals had dispersed evenly throughout the pollen tube (Fig. 1A.c). During a 2-h co-culture with FL-ODN, most pollen tubes showed a similar distribution pattern of fluorescent signal (Fig. S1). These results indicate that the ODNs could effectively enter the pollen tubes within a short period. To determine whether ODN uptake into pollen tubes occurs via endocytosis, we used FM4-64 to track endosome move.
