Movement of Lipid Droplets in the Arabidopsis Pollen Tube Is Dependent on the Actomyosin System

Abstract

The growth of pollen tubes, which depends on actin filaments, is pivotal for plant reproduction. Pharmacological experiments showed that while oryzalin and brefeldin A treatments had no significant effect on the lipid droplets (LDs) trafficking, while 2,3-butanedione monoxime (BDM), latrunculin B, SMIFH2, and cytochalasin D treatments slowed down LDs trafficking, in such a manner that only residual wobbling was observed, suggesting that trafficking of LDs in pollen tube is related to F-actin. While the trafficking of LDs in the wild-type pollen tubes and in myo11-2, myo11b1-1, myo11c1-1, and myo11c2-1 single mutants and myo11a1-1/myo11a2-1 double mutant were normal, their trafficking slowed down in a myosin-XI double knockout (myo11c1-1/myo11c2-1) mutant. These observations suggest that Myo11C1 and Myo11C2 motors are involved in LDs movement in pollen tubes, and they share functional redundancy. Hence, LDs movement in Arabidopsis pollen tubes relies on the actomyosin system.

Keywords: Arabidopsis thaliana; actin; lipid droplets; myosin; pollen tube.

Figure 1

Effects of different pharmacological agents on the mobility of LDs in WT pollen tubes. (a) LDs’ movement was imaged at 0 s (red signals) and 6 s (green signals). Some pollen tubes were treated with 10 µM oryzalin, 50 µM brefeldin A, 40 µM cytochalasin D, 100 µM SMIFH2, 3 nM latrunculin B, and 50 mM 2,3-butanedione monoxime (BDM), respectively. LDs in pollen tubes were stained by the neutral lipid stain Nile Red. The 0 s DIC (differential interference contrast) image was chosen as the representative one. 3× enlarged merged images were shown at the right. Scale bar = 2 µm. (b) The cumulative frequency distribution curve of the speed of pollen tubes LDs in 5 min time-lapse images. (c) The mean velocity of lipid droplets movements in pollen tubes treated with 10 µM oryzalin, 50 µM brefeldin A, 40 µM cytochalasin D, 100 µM SMIFH2, 3 nM latrunculin B, and 50 mM 2,3-butanedione monoxime (BDM), for 15 min were compared with that of untreated pollen tubes. The results are expressed as the mean ± standard error; n = 5. “ns” indicates no significant differences between control and treatments. Asterisks indicate significant differences between control and each of BDM, latrunculin B, SMIFH2, and cytochalasin D treatments (**** p < 0.0001).

Figure 2

Movement of LDs in the pollen tubes of myosin mutants and WT plants. (a) LDs’ movement was imaged at 0 s (red signals) and 6 s (green signals) in pollen tubes of WT and myo11a1-1/myo11a2-1, myo11b1-1, myo11c1-1/myo11c2-1, myo11-2, myo11c1-1, and myo11c2-1 mutants. LDs in pollen tubes were stained by the neutral lipid stain Nile Red. The 0 s DIC (differential interference contrast) image was shown at the left. 3x enlarged merged images were shown at the right. Scale bar = 2 µm. (b) The cumulative frequency distribution curve of the speed of LDs imaged in 5 min time-lapse in the pollen tubes of WT and myo11a1-1/myo11a2-1, myo11b1-1, myo11c1-1/myo11c2-1, myo11-2, myo11c1-1, and myo11c2-1 mutants. LDs in myo11c1-1/myo11c2-1 pollen tubes (blue lines) moved more slowly than in WT pollen tubes (red lines). (c) The mean velocity of LDs’ movements in pollen tubes in WT and myo11a1-1/myo11a2-1, myo11b1-1, myo11c1-1/myo11c2-1, myo11-2, myo11c1-1, and myo11c2-1 mutants was analyzed. The results are expressed as the mean ± standard error; n = 5. “ns” indicates no significant differences between WT and each of the myo11a1-1/myo11a2-1, myo11b1-1, myo11-2, myo11c1-1, and myo11c2-1 mutants. Asterisks indicate significant differences between WT and myo11c1-1/myo11c2-1 (**** p < 0.0001).